Functional MRI (fMRI) studies have shown increased activation of ipsilateral motor areas during hand movement in patients with multiple sclerosis (MS). We hypothesized that these changes could be due to disruption of transcallosal inhibitory pathways. We studied 18 patients with relapsing-remitting MS. Conventional T1- and T2-weighted images were acquired and lesion load (LL) measured. Diffusion tensor imaging (DTI) was performed to estimate fractional anisotropy (FA) and mean diffusivity (MD) in the body of the corpus callosum (CC). fMRI was obtained during a right-hand motor task. Patients were studied to evaluate transcallosal inhibition (TCI, latency and duration) and central conduction time (CCT). Eighteen normal subjects were studied with the same techniques. Patients showed increased MD (P < 0.0005) and reduced FA (P < 0.0005) in the body of the CC. Mean latency and duration of TCI were altered in 12 patients and absent in the others. Between-group analysis showed greater activation in patients in bilateral premotor, primary motor (M1), and middle cingulate cortices and in the ipsilateral supplementary motor area, insula, and thalamus. A multivariate analysis between activation patterns, structural MRI, and neurophysiological findings demonstrated positive correlations between T1-LL, MD in the body of CC, and activation of the ipsilateral motor cortex (iM1) in patients. Duration of TCI was negatively correlated with activation in the iM1. Our data suggest that functional changes in iM1 in patients with MS during a motor task partially represents a consequence of loss of transcallosal inhibitory fibers. Hum Brain Mapp, 2006. © 2006 Wiley-Liss, Inc.