Two groups of retinal cone bipolar cells (CBCs) in rats were found to express voltage-gated Na+ channels. The axon terminals of the first group stratify in sublamina 2 of the inner plexiform layer (IPL) and partially overlap with the OFF-cholinergic band. This group was identified as type 3 CBCs. The axon terminals of the second group stratify in sublamina 3 of the IPL, slightly distal to the ON-cholinergic band. Cells of this second group resemble type 5 CBCs. In addition, we observed another group of ON-type CBCs with terminal stratification similar to that of the second group. However, this latter group did not show any Na+ current, instead exhibiting a large hyperpolarization-activated cyclic nucleotide-gated cation current, suggesting the existence of two subclasses of physiologically distinct type 5 CBCs. Both groups of Na+-expressing bipolar cells were capable of generating a rapid tetrodotoxin-sensitive action potential as revealed by current injection. Multiple spike-like potentials were also observed in some of these cells. Results of this study provide valuable insights into the function of voltage-gated Na+ channels of retinal bipolar cells in retinal processing.