Area of the midsagittal section of the corpus callosum, particularly in the region of the isthmus, was found previously to be greater in non-consistent-right-handed than consistent-right-handed men in a sample of 15 postmortem cases. Seven cases were obtained subsequent to this analysis. The new cases showed the same association previously observed between hand preference and area of the corpus callosum and its isthmus. In addition, a high negative correlation was found between isthmal area and a quantitative score of the direction and magnitude of hand preference. In the new cases, handedness was predicted better than chance using statistical functions of callosal anatomy derived from the previous group of 15 cases. These results support a relationship between variation in callosal anatomy and handedness in men and the hypothesis of a relationship between callosal morphology and functional asymmetry. The lack of such a relationship among women suggests that the developmental mechanisms leading to callosal anatomical variation in relation to lateralization are influenced by sex hormones. Methodological issues in the use of magnetic resonance imaging for the quantitative study of callosal anatomy are discussed.