The mechanism of down-regulation of L-type Ca2+ channel (L-VOC) was investigated in rat aortic smooth muscle cells in primary culture. On culture days 3–5, the cells actively incorporated the 5-bromo-2′-deoxy-uridine (BrdU), and did not respond to K+ depolarization nor express α1C subunit of L-VOC. At confluence on day 8, BrdU incorporation decreased, and the cells up-regulated α1C subunit mRNA, expressed α1C subunit protein at cell periphery, and responded to K+ depolarization. Treating the proliferating cells on day 3 with serum-free media or 10 μM PD98059, a MAP kinase kinase inhibitor, for 2 days induced the expression of α1C subunit protein and the responsiveness to K+ depolarization. However, the serum starvation, but not PD98059, decreased the BrdU incorporation and increased the α1C subunit mRNA. It is concluded that the expression of L-VOC is substantially suppressed in the proliferating cells due to two mechanisms; a MAP kinase-mediated post-transcriptional down-regulation and the transcriptional down-regulation by additional mitogenic signals. J. Cell. Biochem. 87: 242–251, 2002. © 2002 Wiley-Liss, Inc.