The aim of the present study was to examine whether handedness and gender are associated with microstructural differences in human corpus callosum (CC). For this purpose, diffusion-tensor magnetic resonance imaging (DTI) was performed on 34 right- and 33 left-handed subjects of both sexes. Four quantitative variables (Mean Diffusion, Relative Anisotropy, parallel and orthogonal diffusion) were computed within the CC. A significantly increased anisotropy was found in left- as compared to right-handed subjects, and in men as compared to women. Additionally, both overall and orthogonal diffusion were significantly lower in left- than in right-handed subjects. Possible interpretations of these findings are discussed. The novel DTI technique promises to further advance current understanding of morphological structure in the living brain.