Magnetization transfer imaging is an MRI technique that provides quantitative information about in vivo tissue integrity, including myelin and axonal content, and is expressed as the magnetization transfer ratio (MTR). The optic neuritis lesion can model the MS lesion in vivo and permits use of non-invasive markers of optic nerve myelination (visual evoked potential [VEP] latency) and retinal neuroaxonal loss (optical coherence tomography [OCT]) to provide further information about the in vivo substrates of optic nerve MTR. Twenty-five patients with optic neuritis were studied using an optic nerve MTR sequence, quantitative visual function testing, VEPs and OCT, along with 15 controls. MTR was reduced in affected nerves compared to both clinically unaffected nerves from patients and control nerves (P < 0.001). Whole-nerve MTR correlated modestly with central-field VEP latency but more strongly when lesion-only MTR was measured, when a modest correlation with whole-field VEP latency emerged. OCT-quantified retinal neuroaxonal loss also correlated with MTR. In conclusion, markers of optic nerve myelination and axonal loss both correlate with optic nerve MTR. Because axonal loss following optic neuritis also results in myelin loss, the relative contributions of the two pathological conditions to the MTR measures cannot be estimated from this study.