—Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein–coupled receptors, termed A₁, A_2A, A_2B, and A₃. We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A₁ agonist N⁶-cyclopentyladenosine nor the A_2A agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A_2A antagonist SCH58261 or the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A_2B adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A_2B adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A_2B adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.