The tritiated amino acid autoradiographic method was employed to characterize the patterns of commissural projections originating in the hippocampus of the rat, guinea pig, rabbit, and cat. The results demonstrate that significant differences between species are present despite the overall similarity of the projections. In the rat and cat the commissural connections are widely distributed along the septotemporal axis of the hippocampus, but in the guinea pig and rabbit they are less widely distributed along this axis. Second, within the hippocampus proper the radial distribution of the commissural projection is species specific. In the rat, CA3 commissural projections are present in both the strata oriens and radiatum, but the densest projection is to the stratum oriens. In the guinea pig the radial distribution of this projection is similar to that observed in the rat, but in the rabbit the projection is almost entirely confined to the stratum oriens. In contrast, in the cat the CA3 commissural projection is very dense to the stratum radiatum and sparse to stratum oriens. An analysis of the relative density of label in the molecular layer of the fascia dentata suggests that the density of the commissural projection from CA4 is much greater in the rat and cat than in the guinea pig or rabbit. These results indicate that care must be exercised in the generalization of connectional data between species. The results also suggest a possible explanation for differences observed in the electrophysiology of these connections between species. Copyright © 1988 Alan R. Liss, Inc.