Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO) via the catabolism of heme by the enzyme heme oxygenase (HO). In the present study, we found that serum stimulated a time- and concentration-dependent increase in the levels of HO-1 messenger RNA (mRNA) and protein in vascular SMCs. The induction of HO-1 expression by serum was inhibited by actinomycin D or cycloheximide. In addition, serum stimulated HO activity, as reflected by an increase in the concentration of bilirubin in the culture media. Treatment of vascular SMCs with serum stimulated DNA synthesis and this was potentiated by the HO inhibitors, zinc and tin protoporphyrin-IX as well as by the CO scavenger, hemoglobin. The iron chelator desferrioxamine had no effect on DNA synthesis. However, exposure of vascular SMCs to exogenous CO inhibited serum-stimulated SMC proliferation and the phosphorylation of retinoblastoma protein. In addition, CO arrested SMCs at the G(1)/S transition phase of the cell cycle and selectively blocked the serum-stimulated expression of cyclin A mRNA and protein without affecting the expression of cyclin D1 and E. CO also inhibited the serum-stimulated activation of cyclin A-associated kinase activity and cyclin-dependent kinase 2 activity. These results demonstrate that serum stimulates HO-1 gene expression and CO synthesis. Furthermore, they show that CO acts in a negative feedback fashion to inhibit vascular SMC growth by regulating specific components of the cell cycle machinery. The capacity of vascular mitogens to induce CO synthesis may provide a novel mechanism by which these agents modulate cell growth.