Judgment of abnormalities in fetal cortical axon development is more sensitive when a good standard of normal ontogeny is established. The recent availability of postmortem tract tracing methods has greatly improved the observation of axon extension and growth cone morphology in mouse fetuses, which allows much stronger statements about the timing of crucial steps in the formation of the corpus callosum in particular. The first outgrowth and crossing of midplane by axons of the corpus callosum (CC) were examined in 153 normal mouse embryos and fetuses of the hybrid cross B6D2F2/J with carbocyanine dyes applied to brains fixed by perfusion. In most brains a crystal of DiI was inserted into either frontal, parietal, temporal, or occipital cortex in one hemisphere, and a crystal of DiA was placed into a different site in the opposite hemisphere. Although dye diffusion obscured the emergence of axons, linear regression analysis revealed that the first callosal axons emerged from their cortical cells of origin at about 0.4 g body weight or 15.5 days after conception for all four sites. Subsequent axon growth rate was substantially faster for those from frontal cortex (3.2 mm/day) than occipital cortex (1.8 mm/day). Axons from frontal cortex crossed the cerebral midplane first (0.69 g, E16.3), followed by those from parietal (0.74 g), temporal (0.77 g) and occipital cortex (0.92 g, E16.9). Prior to crossing midplane, the pioneering CC axons were usually 200 μm or less in advance of the main bundle, but when they crossed midplane and encountered CC axons growing from homotopic sites in the opposite hemisphere, the pioneering axons were often 0.5 to 2.5 mm ahead of the main bundle. Growth cones were usually large and complex until they had crossed midplane and were thereafter smaller with simple and flat morphologies. The topography of axons in the CC at midplane was organized according to cortical region of origin from the very beginning, when the CC was only a small cap over the hippocampal commissure and dorsal septum. The quantitative results provide a convenient, standard for normal callosal development in mice and should facilitate comparative studies. © 1992 Wiley-Liss, Inc.