Multi-modal imaging has synergistic effects not only to improve image quality and accuracy in quantification, but also to understand complex in vivo metabolic processes^1,2. In this study, we present our initial experiences of simultaneous in vivo metabolic imaging using ¹H MRI, Time-Of-Flight (TOF) ¹⁸F-FDG PET, and hyperpolarized ¹³C-pyruvate MRSI in rat glioma models.Two models of rat glioma were imaged using ¹H MRI, ¹⁸F-FDG PET, and hyperpolarized ¹³C MRSI: male Wistar rats implanted with C6 glioma (N = 6, 260-393g, 10 days after C6 implantation) and male Wistar rats with N-Ethyl-N-nitrosourea (ENU)-induced glioma (N = 2, 383-444g, 116-123 days old). Each rat was anesthetized and catheterized in the tail vein before placing at the center of the GE 3T Signa PET/MR scanner. The imaging session started with a bolus injection of 1-mCi ¹⁸F-FDG (diluted to be 1ml), immediately followed by 1.5hrs of TOF PET acquisition. While PET data were collected proton MR images were acquired simultaneously using MRAC (for subject tissue attenuation correction), 2D T₂-weighted fast spin echo (FSE), 3D T₂-weighted CUBE, fluid-attenuated inversion recovery (FLAIR), and diffusion-weighed imaging (DWI) sequences. ¹³C MRSI data were also acquired during the PET scan after a bolus injection of 80-mM [1-¹³C]pyruvate, which was polarized for ~5hrs using GE SPINlab, using two imaging sequences: single time-point volumetric spiral chemical shift imaging³ (CSI, four spatial interleaves, acquisition time = 5.5s, field of view (FOV) = 64×64×48mm³, nominal spatial resolution = 4×4×4mm³), dynamic 2D spiral CSI⁴ (four spatial interleaves, temporal resolution = 3s, 20 time points, FOV = 64×64mm², nominal spatial resolution = 4×4mm², variable flip angle). Before the imaging session ended, T₁-weighted spin echo (SE) images were obtained before and after Gadolinium injection (0.9ml, Gd:saline=1:2). The overall protocol is summarized in Figure 1. ¹H-¹³C dual-tuned quadrature coil (Ø=80mm) was used for the MR(S)I, but the coil attenuation was not taken into account for the PET reconstruction. The dynamic PET data was reconstructed in 30-s bins (0-20min) and in 2-min bins (20-90min) using a fully 3D TOF iterative ordered subsets expectation maximization (OSEM) algorithm (24 subsets, 3 iterations, timing resolution=400ps). All the glioma rats were fasted for 20-24 hrs prior to ¹⁸F-FDG injection. Moreover, to investigate the effect of 80-mM pyruvate bolus injection on FDG uptake, three of the C6 glioma rats were scanned twice: ~1min after saline vs. pyruvate injections on two consecutive days (20hrs of fasting for both days). Temperature and respiration were maintained at ~36^oC and 60 breaths/min, respectively, throughout the experiment. Three tumor-bearing axial slices from a representative ENU-induced glioma rat are shown in Figure 2. The tumor region was confirmed with ¹H MRI and showed upregulated ¹⁸F-FDG uptake and increased ¹³C-lactate labeling. Dynamic ¹⁸F-FDG uptake with a temporal resolution of 30s and dynamic imaging of [1-¹³C]lactate and [1-¹³C]pyruvate with a temporal resolution of 3s (Fig. 3) were reconstructed with sufficient SNR. Whereas the ¹³C-lactate production was consistently higher in the tumor ROI than in normal-appearing ROI, FDG-uptake curves in the brain showed two regimes: an initial fast uptake regime and a slowly increasing regime. The glioma ROIs tended to have slightly lower FDG-uptake in the first regime and more uptake in the second regime than the normal-appearing brain ROIs (Fig. 4A). The rats, infused with an additional 80-mM pyruvate prior to ¹⁸F-FDG injection, showed slower uptake rate of ¹⁸F-FDG in normal-appearing brain (11.8 ± 1.6 Bq/ml/s) in the second regime compared to the PET data acquired after saline injection (8.5 ± 0.6 Bq/ml/s, P = 0.04, Fig. 4). Accordingly, the increase in image contrast between tumor and normal-appearing brain was observed in all the rats (4.35 % at baseline, 9.60 % with pyruvate, P = 0.016). The change of FDG kinetics due to pyruvate injection suggests that FDG uptake via the glucose transporter (GLUT) and pyruvate uptake via the monocarboxylic transporter (MCT) might be competing processes.We demonstrated the feasibility of simultaneous investigation of in vivo metabolism using ¹H MRI, ¹⁸F-FDG PET, and hyperpolarized ¹³C-pyruvate MRSI in both C6 xenograft and ENU-induced brain tumor models.