Cerebral blood flow (CBF) measurements are critical to assessment of many cerebrovascular disorders including stroke. In the past 15 years, arterial spin labeling (ASL) MRI has gained traction as a promising, noninvasive way to quantify CBF [1]. However, ASL is not routinely used in the clinic because it lacks validation. Most validation studies to date have compared ASL to the [15O]-water PET reference standard in separate imaging sessions, such that natural CBF fluctuations due to different physiological states, diet, and diurnal cycles confound the comparison [2, 3]. This study performs a head-to-head comparison between pseudo-continuous ASL and [15O]-water PET reference scans collected simultaneously on a hybrid PET-MRI system. We aim (1) to compare the CBF response of the brain to Diamox as measured by PET and by MRI; and (2) to evaluate the scan-rescan reproducibility of CBF measurements by each image modality. We also discuss future opportunities in using image-based arterial input functions (AIF) from the hybrid system to improve CBF quantification.Five healthy volunteers (two female; ages 24-40 years) were scanned on a 3T time of flight PET-MRI hybrid system (GE Healthcare, Milwaukee, WI). PET imaging with 15-25 mCi of [15O]-water was performed before and after administration of 15mg/kg of Diamox, which increases CBF. PET scans commenced immediately after each tracer injection and coincided with ASL MRI scans. Four subjects received successive repeat PET scans and all subjects received repeat ASL scans before Diamox. Relative maps of CBF were created from integration of PET counts over the first 2 minutes after tracer injection and normalization for tracer dose. The ASL scan parameters included pseudo-continuous labeling with label duration of 1500ms and post-label delay of 2025ms; TR/TE = 4850/10.7 ms; slice thickness = 4mm; bandwidth = 62.5kHz; and spiral readout (8 arms of 512 samples). Quantitative CBF maps were calculated for using consensus values for the longitudinal relaxation of arterial blood and tag efficiency [4]. All perfusion images were registered to a common stereotactic space (the Montreal Neurological Institute template) with FSL software. Scan-rescan reproducibility of relative CBF by PET and of absolute CBF by ASL was assessed using coefficient of variation (COV) = standard deviation of the repeat scans / mean between the scans. Group averages of relative CBF by PET and baseline CBF by ASL showed similar spatial distribution across the 5 volunteers (Figure 1). Absolute gray matter CBF measured by ASL increased from 48.9±8 ml/100g/min at rest to 68.1±12 ml/100g/min after Diamox administration. For the same volunteer, we assumed a similar AIF following each tracer injection, such that comparing PET images before and after Diamox revealed the relative CBF augmentation (after accounting for tracer dose). Both modalities revealed comparable CBF augmentation in the gray and white matter due to Diamox (Figure 2), with slightly higher CBF increase of 44.9% by ASL and 39.9% by PET. COV maps of scan-rescan reproducibility for each image modality are shown averaged over all subjects (Figure 3). The reproducibility of successive scans was similar for PET and MRI; ASL showed slightly lower COV (11.5%) across the gray matter compared to PET (14.6%). The COV maps tended to be higher for white matter, which is expected due to the low perfusion signal in those regions.Hybrid PET-MRI allows simultaneous observations of the same brain perfusion state by ASL MRI and the [15O]-water PET reference standard for validation. Our initial findings suggest that ASL can reliably detect increases in CBF during Diamox administration consistent with PET measurements and with lower COV. In future work, we will focus on kinetic modeling of quantitative CBF from [15O]-water PET. This analysis can potentially leverage hybrid PET-MRI information by identifying image-based AIFs from regions of interest within carotid arteries on co-localized, high-resolution MRI angiograms. Knowledge of the artery location from high-resolution MRI can be used to mitigate spill-over effects that contaminate the AIF peak shape and height, and thus absolute CBF values. Furthermore, AIFs determined at the carotid may be a more accurate representation of tracer arrival to the brain. For instanced, the [15O]-water AIF may have a higher peak and arrive more quickly to the brain carotids after Diamox compared to baseline (Figure 4). This difference in AIF would not have been detected from traditional arterial sampling at a radial artery of the arm. Thus, simultaneous PET-MRI offers dual information that can help to validate ASL as a clinical and scientific tool to study brain perfusion, and to improve its measurement by both modalities.