The diffusion properties of water are sensitive to microscopic changes in the white matter of multiple sclerosis (MS) patients. Typical MRI measures of disease burden in MS demonstrate modest to poor correlation with disability. Functional MRI and DTI-based fiber tracking were used to define the interhemispheric white matter pathway connecting bilateral supplementary motor areas (SMA) in 16 MS patients sand 16 control subjects. Fractional anisotropy (FA), mean diffusivity (MD), longitudinal (lambda(1)) and transverse diffusivity (lambda(2)) were measured along this pathway in all subjects. Mean FA was 0.587 +/- 0.032 for patients and 0.608 +/- 0.020 for controls (P < 0.02). Mean MD was (0.821 +/- 0.055) x 10(-3) mm(2) s(-1) for patients and (0.770 +/- 0.020) x 10(-3) mm(2) s(-1) for controls (P < 0.004). Mean lambda(1) values were (1.462 +/- 0.099) x 10(-3) mm(2) s(-1) for patients and (1.400 +/- 0.034) x 10(-3) mm(2) s(-1) for controls (P < 0.02). Mean lambda(2) values were (0.500 +/- 0.047) x 10(-3) mm(2) s(-1) for patients and (0.454 +/- 0.027) x 10(-3) mm(2) s(-1) for controls (P < 0.001). In addition, the correlation between the Multiple Sclerosis Functional Composite (MSFC) and transverse diffusivity was -0.341 (P < 0.05). The component test of the MSFC most related to the SMA pathway studied with our MRI method (Nine-hole Peg Test) showed significant correlation with transverse diffusivity (r = 0.392, P < 0.02), indicating that probing functional pathways with MRI measures can lead to a better reflection of disease status.