Voltage-dependent Ca²⁺ channels (VDCCs), which consist of multiple subtypes, regulate vascular tone in developing arterial smooth muscle, including the ductus arteriosus (DA). First, we examined the expression of VDCC subunits in the Wistar rat DA during development. Among α₁-subunits, α_1C and α_1G were the most predominant isoforms. Maternal administration of vitamin A significantly increased α_1C- and α_1G-transcripts. Second, we examined the effect of VDCC subunits on proliferation of DA smooth muscle cells. We found that 1 μM nitrendipine (an L-type Ca²⁺ channel blocker) and kurtoxin (a T-type Ca²⁺ channel blocker) significantly decreased [³H]thymidine incorporation and that 3 μM efonidipine (an L- and T-type Ca²⁺ channel blocker) further decreased [³H]thymidine incorporation, suggesting that L- and T-type Ca²⁺ channels are involved in smooth muscle cell proliferation in the DA. Third, we found that a novel alternatively spliced variant of the α_1C-isoform was highly expressed in the neointimal cushion of the DA, where proliferating and migrating smooth muscle cells are abundant. The basic channel properties of the spliced variant did not differ from those of the conventional α_1C-subunit. We conclude that multiple VDCC subunits were identified in the DA, and, in particular, α_1C- and α_1G-subunits were predominant in the DA. A novel spliced variant of the α_1C-subunit gene may play a distinct role in neointimal cushion formation in the DA.