This study examined the contribution of cAMP signaling to the modulation of vascular smooth muscle cell (VSMC) proliferation by adenosine. At a concentration of 1 mM, adenosine inhibited [3H]-thymidine uptake, measured as the initial rate of isotope influx, by 10-fold. Diminution of [3H]-thymidine uptake by adenosine was independent of the presence of A1- and A2-receptor antagonists, indicating that adenosine competes with thymidine for plasma membrane transporter-binding sites. Considering these results, in order to estimate [3H]-thymidine DNA labeling, VSMCs were preincubated with adenosine for 48 h, and adenosine was then omitted during the subsequent 2 h of incubation in [3H]-thymidine-containing medium. In serum-depleted VSMCs, preincubation with 100 µM or 1,000 µM adenosine augmented DNA synthesis by approximately 6- and 3-fold, respectively, whereas the increment of DNA synthesis triggered by serum was decreased in the presence of adenosine by 20–30%. Both cAMP production and inhibition of DNA synthesis by adenosine in serum-supplied cells were independent of the presence of the A1-antagonist 1,2-dipropyl-8-cyclopentylxanthine (DPCPX), but were abolished by the A2-antagonist 1,3-dimethyl-7-propylxanthine (DMPX). In contrast, the activation of DNA synthesis in serum-depleted cells by adenosine was decreased in the presence of DPCPX and DMPX by approximately 30 and 40%, respectively. Both in serum-supplied and -depleted VSMCs, dose-dependent elevation of cAMP production with an adenylate cyclase activator, forskolin, reduced DNA synthesis by up to 40–60%. Thus, our results show that in addition to suppressing thymidine uptake, adenosine depresses the DNA synthesis triggered by serum-derived growth factors and stimulates DNA synthesis in serum-depleted cells. These data also suggest that the inhibition of DNA synthesis is mediated by cAMP production where the activation of DNA synthesis is independent of cAMP signaling.