A precise description of the timing and route traveled by axons traversing the telencephalic midline through the ventral hippocampal commissure (HC) is essential for understanding the role it plays in the formation of the corpus callosum (CC). A normal baseline of HC development was described in B6D2F2 hybrid mice and then compared with two inbred strains of mice displaying callosal agenesis, BALB/cWah1 (50% CC defect) and 129/J (70% CC defect), their F2 hybrid (C129F2—33% CC defect), and a recombinant inbred strain (Rl‐1—100% CC defect) derived from pairs of C129F2 mice. Embryos weighing from 0.25 g to 0.70 g (E14.5–E17) were collected and fixed by perfusion. Axon tracts were labeled using crystals of the lipophilic dyes Dil and DiA inserted into the hippocampal fimbria and cerebral cortex. HC axons in B6D2F2 mice first cross the midline at about 0.350 g body weight (E14.8) by traveling over the dorsal septum and along the pia membrane lining the longitudinal fissure. Earlier crossing was prevented by the presence of a deep cleft formed by the longitudinal fissure extending down into the septal region. Subsequent axons fasciculated along existing axons, gradually building the dorsoventral height of the HC to about 200 μm by 0.600 g. The earliest callosal axons from frontal cortex crossed the midline at 0.620 g and were clearly seen fasciculating along and between existing hippocampal axons at the dorsal surface of the HC as they crossed. In the acallosal strains, HC formation was delayed by the continued presence of the cleft deep in the septal region. This delay in time of crossing was correlated with later CC defect expression. Initial HC crossing occurred at about 0.470 g (E16.25) in BALB mice and about 0.520 g (E16.5) in 129 mice. In the Rl‐1 embryos, first HC crossing was estimated at about 0.750 g (E17.5), although several older embryos showed no crossing. These results show the importance of the HC for successful CC formation and suggest that absent CC arises as a consequence of a developmental defect which affects the formation of the hippocampal commissure prior to arrival of CC axons at midplane. Hippocampus 7:2–14, 1997. © 1997 Wiley‐Liss, Inc.