Magnetization Transfer (MT) contrast has been used to study brain myelination and the characterization of myelin disorders (1–3). Most MT studies ignore potential chemical shifts between macromolecular protons (MPs) and water protons (WPs) (1–4), despite reported evidence to the contrary (5,6). Ignoring chemical shift results in errors in estimation of MT parameters, especially at high field. Experiments with steady state off-resonance RF irradiation have been performed to characterize MP chemical shift and its effect on MT, however these generally suffer from confounds related to direct WP saturation and CEST effects. Here we propose a novel approach based on a delay-dependent pulsed MT experiment.Our approach to measure the effect of chemical shift between MP and WP on MT is based on a 2-pool model of MT and involved two steps: 1) The measurement of 2-pool model parameters based on fitting of WP signal at various delay times $$$\Delta$$$ following MP saturation with a 4 ms composite MT pulse. 2) The use of these parameters to calculate MP saturation levels from the measurement of delay-dependent WP saturation after a 12 ms hyperbolic secant (HS) pulse at various off-resonance frequencies. The composite pulse was 4ms long and consisted of variable number of subpulses and different pulse amplitudes: B1=250Hz with 2 subpulses, 500Hz with 4, 1000Hz with 8, 2000Hz with 16, 4000Hz with 32 , 5000Hz with 32, with flip angles of 0.5x, -x, x …, -x, 0.5x. $$$\Delta$$$ ranged from 5.8 to 1600ms. HS pulse duration was 12ms and peak B1 amplitude was 500Hz; $$$\Delta$$$ ranged from 6 to 406ms, and frequency offset ranged from -30kHz to 30kHz. A fixed common marmoset brain was scanned on a Bruker 4.7T scanner. Multi-gradient echo was used for acquisition, with 0.25mm isotropic resolution, TE of 3ms, TR of 3s, and nominal excitation flip angle of 90°. Reference signal level was measured by omitting the MT pulse. Regions of interest (ROI’s) were selected in white matter (WM) and grey matter (GM). The averaged signal amplitude $$$S(\Delta)$$$ in these ROIs was converted into saturation fraction $$$Sat_w(\Delta)=1-S(\Delta)/S_{ref}$$$ and fitted to: $$$Sat_{w}(\Delta)=A_{1}e^{-\lambda_1 \Delta}+A_{2}e^{-\lambda_2 \Delta}$$$, where $$$\lambda_{1,2}= \frac{1}{2}(k_{wm}+R_{1w}+k_{mw}+R_{1m} \pm \sqrt{ (k_{wm}+R_{1w}-k_{mw}-R_{1m})^{2}+4k_{mw}k_{wm} })$$$, subscripts $$$w$$$ and $$$m$$$ refer to water and macromolecular protons respectively, $$$k_{mw}$$$ is the forward exchange rate, and $$$k_{wm}=k_{mw}f/(1-f)$$$ the reverse rate, with $$$f$$$ referring to MP fraction. $$$R_{1m}$$$ and $$$R_{1w}$$$ were assumed to be uniform across WM and GM. MP saturation was calculated from: $$$Sat_m(0)=A_{1}(-\lambda_{1}+k_{wm}+R_{1w})/k_{wm}+A_{2}(-\lambda_{2}+k_{wm}+R_{1w})/k_{wm}$$$. The resonance frequency of the MP was determined from the MP saturation as function of offset frequency of the HS pulse by fitting this data with a super-Lorentzian line shape. While this method cannot be used to measure the line shape, it does allow estimation of the (average) MP resonance frequency.Figure 1 shows the fitting of WP saturation following a composite MT pulse with varying pulse amplitudes. WP saturation leveled off at 2000 Hz pulse amplitude suggesting full (100%) MP saturation. Figure 2 shows the extracted 2-pool model parameters. MP saturation dependence on off-resonance frequency (Fig. 3) shows a maximum at -540 Hz (-2.7 ppm) for marmoset, in both WM and GM. Preliminary results from white matters in human subjects at 7T showed a -2.56ppm offset.The pulses MT experiment allowed investigation of MT asymmetries without confounding effects from direct WP saturation and CEST. The former was effectively accounted for in the 2-pool fitting approach, whereas the latter was avoided due to the short duration of the MT pulse. Based on this, the MT effect was found to be symmetric around -2.72 ppm from water, which is consistent with previous work (5) and the range of chemical shift of alkyl protons, which are abundant in membrane lipids and constitute a major fraction of MPs. The relatively low power of the pulse MT approach make it suitable to be applied $$$in$$$ $$$vivo$$$, even at high field in humans. The observed MP chemical shift should be accounted for in the analysis of MT experiments that aim at quantifying parameters such as MP pool fraction ($$$f$$$) and MT exchange rates ($$$k$$$).