Retinal bipolar cells make up a class of at least 11 distinct interneurons that have been classified through morphological and molecular approaches. Previous work has shown that the paired-like homeodomain transcription factor Vsx1 is essential for the proper development of a subset of these interneurons. In Vsx1-null mice, bipolar cells are properly specified but exhibit terminal differentiation defects characterized by reduced expression of OFF bipolar cell markers and defects in OFF visual signaling. Here, we further examined the role of Vsx1 in OFF bipolar cells using recently identified cell-type-specific markers. In contrast to its previously characterized expression in type 2 OFF bipolar cells, Vsx1 expression was not detected in type 3 OFF bipolar cells, by either immunohistological or transgenic reporter labeling approaches. This observation was unexpected given previous findings that Cabp5 immunolabeling of type 3 bipolar cell axon terminals is reduced in Vsx1-null mice. However, we observed reduced levels of the type 3a bipolar cell marker hyperpolarization-activated and cyclic nucleotide-gated channel 4 (HCN4) in Vsx1-null mice, which is consistent with a requirement for Vsx1 in type 3 bipolar cell differentiation. In contrast, expression of the type 3b bipolar cell marker regulatory subunit RII-beta of protein kinase A was unchanged. Despite the absence of Vsx1 in mature type 3 bipolar cells, colabeling of Vsx1 and HCN4 was observed at postnatal stages. These findings reveal a role for Vsx1 in type 3a bipolar cells and suggest that Vsx1 function is required transiently in this cell type during the postnatal period. J. Comp. Neurol., 2012. © 2011 Wiley Periodicals, Inc.