This study investigated the mechanisms by which adenosine stimulates proliferation of microvascular endothelial cells. The metabolic byproducts of adenosine, inosine and hypoxanthine were unable to stimulate proliferation. When adenosine uptake was prevented, the stimulation of proliferation was unchanged, suggesting that uptake of adenosine with subsequent incorporation into the nucleotide pool is not the mechanism for increasing proliferation. Treatment of endothelial cells with adenosine analogues, presumably selective for either the A1 or A2 receptor, stimulated proliferation equally. This suggested that adenosine 3', 5'-cyclic monophosphate (cAMP) might not mediate the proliferative response to adenosine. However, radioimmunoassay of cell extracts after treatment with either analogue showed an increase in cAMP. In addition, adenylate cyclase blockade with 2', 5'-dideoxyadenosine prevented the proliferative response brought about by these analogues. These data suggest that the proliferative response to adenosine depends on an increase in cAMP. A 2-h pulse of cholera toxin stimulated endothelial cell proliferation, further supporting a role for cAMP. Pretreatment of endothelial cells with pertussis toxin blocked the stimulation of proliferation, indicating that a Gi or similar G protein is also involved in proliferation. We conclude that the proliferative response to adenosine involves a pertussis toxin-sensitive substrate as well as an increase in cAMP.