The anatomy of the corpus callosum has received renewed interest during recent years due to the increasing number of callosotomies performed to treat intraventricular lesions, as well as some forms of generalized epilepsy. We have previously reported on the microsurgical anatomy of the corpus callosum and identified specific anatomical reference landmarks that can be used during surgery. In the present study we have continued the anatomical aspect of this earlier work in a larger number of cases, with in vitro observations (brain out of skull) being compared with the corresponding in vivo features seen in sagittal MRI slices. Fifty-three in vitro microsurgical callosotomies was performed and the data collected compared with a series of 57 in vivo normal MR callosal images. Callosal dimensions were measured on both the anatomical and MRI material, thus overcoming the problems associated with in vitro callosal deformation. Of the anatomical landmarks studied the distance from the genu of the corpus callosum to the bifurcation of the columns of the fornix was found to be useful for the intraoperative evaluation of the extent of rostral callosotomy, as it is not significantly changed in in vitro. The main microsurgical features of rostral callosotomy are presented.