PURPOSE: To investigate the relationship of myelin content, axonal density, and gliosis with the fraction of macromolecular protons (fB) and T2 relaxation of the macromolecular pool (T2B) acquired using quantitative magnetization transfer (qMT) MRI in postmortem brains of subjects with multiple sclerosis (MS). MATERIALS AND METHODS: fB and T2B were acquired in unfixed postmortem brain slices of 20 subjects with MS. The myelin content, axonal count, and severity of gliosis were all quantified histologically. t-Tests and multiple regression were used for analysis. RESULTS: MR indices obtained in unfixed postmortem MS brains were consistent with in vivo values reported in the literature. A significant correlation was detected between Tr(myelin) (inversely proportional to myelin content) and 1) fB (r = -0.80, P < 0.001) and 2) axonal count (r = -0.79, P < 0.001). fB differed between 1) normal-appearing white matter (NAWM) and remyelinated WM lesions (rWMLs) (mean: fB 6.9 [SD 2] vs. 4.0 [1.8], P = 0.01), and 2) rWMLs and demyelinated WMLs (mean: 4.2 [2.2] vs. 2.5 [1.3], P = 0.016). No association was detected between T2B and any of the histological measures. CONCLUSION: fB in MS WM is dependent on myelin content and may be a tool to monitor patients with this condition.