The corpus callosum (CC) represents the major commissural tract connecting the two cerebral hemispheres and is supposed to play crucial integrative role in functional hemispheric specialization. The present study examined whether interindividual variations in macro- and microstructure of the human CC are associated with handedness and gender. Therefore, a combined diffusion-tensor (DTI) and high-resolution morphological MRI study was performed on 34 right- and 33 left-handed subjects of both sexes. The mid-sagittal surface areas and quantitative measures of molecular diffusion (relative anisotropy, mean diffusion) of the total CC and its subregions (genu, truncus, posterior third) were determined. Analysis revealed a larger total callosal area in right- as compared to left-handed subjects and in males as compared to females. Throughout all callosal subregions, anisotropy was found to be increased in left-handed as well as in male subjects, while the mean diffusion was diminished only in left-handers. For the posterior third of the CC, a significant negative correlation (r=-0.34) between anisotropy and area was detected in right-handed subjects. Summarized, significant alterations in the molecular diffusion and in the size of the CC with respect to gender and handedness were revealed in the present study. These findings can be interpreted as handedness- and gender-related differences in macro- and microstructure of the callosal pathways. It was demonstrated that the inspection of the callosal microstructure using DTI yields empirical evidence on interhemispheric connectivity that goes well beyond the information revealed by anatomical measurements alone. Thus, DTI has proven to be a useful additional method in cognitive neuroscience.