Adenylate cyclase activity in cultured rat aortic vascular smooth muscle cells showed a linear correlation with the rate of DNA synthesis. When smooth muscle cells were rendered quiescent by shifting them from a serum-supplemented medium to a medium containing low concentrations of plasma, the cells could be stimulated to proliferate by the addition of serum or by addition of a crude preparation of platelet-derived growth factor. DNA synthesis began at 16 hours and was maximal at 24 hours. Prior to synthesis of DNA there was an increase in adenylate cyclase activity with a peak at 12 hours. Adenylate cyclase activity returned to basal level before DNA synthesis began. The increase in adenylate cyclase activity was not blocked by cycloheximide. Adenylate cyclase activity could also be increased by incubating vascular smooth muscle cells with cholera toxin; however, the time course and magnitude of this increase was different from that caused by growth stimulants. Cholera toxin caused a slight increase in DNA synthesis at 16 hours, but was also cytotoxic to smooth muscle cells. An increase in adenylate cyclase activity may be a prerequisite for the progression from G1 to S.