The avian hippocampal formation differs considerably from that of mammals both in terms of position and cytoarchitecture. On the basis of fiber connections in pigeons, however, we previously proposed that the dorsomedial subdivision (DM) and the V-shaped layer of the hippocampal formation correspond to Ammon's horn and the dentate gyrus of mammals, respectively. In the present study, we provide evidence in support of this hypothesis by double staining hippocampal neurons using tract-tracing and gene expression. After cholera toxin subunit B (CTB) was injected into the lateral septal nucleus (SL), and vesicular glutamate transporter 2 (vGluT2) mRNA, a gene marker for glutamatergic neurons, was visualized in the same retrogradely labeled neurons with in situ hybridization, most CTB+/vGluT2+ neurons were concentrated in DM, but were rare in the V-shaped layer. The distribution pattern of CTB+/vGluT2+ neurons in the hippocampal formation did not change when CTB injection sites were shifted in a rostrocaudal direction in SL. SL expresses a variety of mRNAs for ionotropic glutamate receptor subunits (GluA1, GluA2, GluK2, GluK4, and GluN1). The findings indicate that DM neurons provide descending glutamatergic axons to SL. Additionally, the present study showed that Prox1 mRNA, a gene marker for the dentate gyrus in mammals, was intensely expressed in the V-shaped layer in the pigeon hippocampus. Together these results strengthen our original hypothesis that DM and the V-shaped layer in the pigeon hippocampus are homologous to Ammon's Horn and the dentate gyrus, respectively. 2016 Wiley Periodicals, Inc.