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Direct assessment of myelin has the potential to reveal central nervous system abnormalities and serve as a means to follow patients with demyelinating disorders during treatment. Here, we investigated the feasibility of direct imaging and quantification of the myelin proton pool, without the many possible confounds inherent to indirect methods, via long-T2 suppressed 3D ultra-short echo-time (UTE) and zero echo-time (ZTE) MRI in ovine spinal cord.ZTE and UTE experiments, with and without inversion-recovery (IR) preparation, were conducted in ovine spinal cords before and after D2O exchange of tissue water, on a 9.4T vertical-bore micro-imaging system, along with some feasibility experiments on a 3T whole-body scanner. Myelin density was quantified relative to reference samples containing various mass fractions of purified myelin lipid, extracted via the sucrose gradient extraction technique, and reconstituted by suspension in water, where they spontaneously self-assemble into an ensemble of multi-lamellar liposomes, analogous to native myelin.MR signal amplitudes from reference samples at 9.4T were linearly correlated with myelin concentration (R2 = 0.98–0.99), enabling their use in quantification of myelin fraction in neural tissues. An adiabatic inversion-recovery preparation was found to effectively suppress long-T2 water signal in white matter, leaving short-T2 myelin protons to be imaged. Estimated myelin lipid fractions in white matter were 19.9%–22.5% in the D2O-exchanged spinal cord, and 18.1%–23.5% in the non-exchanged spinal cord. Numerical simulations based on the myelin spectrum suggest that approximately 4.59% of the total myelin proton magnetization is observable by IR-ZTE at 3T due to T2 decay and the inability to excite the shortest T2* components. Approximately 380 μm of point-spread function blurring is predicted, and ZTE images of the spinal cord acquired at 3T were consistent with this estimate.In the present implementation, IR-UTE at 9.4T produced similar estimates of myelin concentration in D2O-exchanged and non-exchanged spinal cord white matter. 3T data suggest that direct myelin imaging is feasible, but remaining challenging on clinical MR systems.9.4T spinal cord myelin density maps show expected contrast between GM and WM.IR-preparation suppression pulses effectively suppress long-T2 signal in white matter.Myelin densities measured in white matter are in the range of 18.1%–23.5%.Myelin density measurements are consistent in non-exchanged and D2O-exchanged cords.Feasibility of direct myelin imaging with ZTE was shown on a 3T clinical scanner.