Adenosine has been reported to stimulate or inhibit the release of angiogenic factors depending on the cell type examined. To test the hypothesis that differential expression of adenosine receptor subtypes contributes to endothelial cell heterogeneity, we studied microvascular (HMEC-1) and umbilical vein (HUVEC) human endothelial cells. Based on mRNA level and stimulation of adenylate cyclase, we found that HUVECs preferentially express A_2A adenosine receptors and HMEC-1 preferentially express A_2B receptors. Neither cells expressed A₁ or A₃ receptors. The nonselective adenosine agonist 5′-N-ethylcarboxamidoadenosine (NECA) increased expression of interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) in HMEC-1, but had no effect in HUVECs. In contrast, the selective A_2A agonist 2-p-(2-carboxyethyl)phenylethylamino-NECA (CGS 21680) had no effect on expression of these angiogenic factors. Cotransfection of each type of adenosine receptors with a luciferase reporter in HMEC-1 showed that A_2B receptors, but not A₁, A_2A, or A₃, activated IL-8 and VEGF promoters. These effects were mimicked by constitutively active αG_q, αG₁₂, and αG₁₃, but not αG_s or αG_i1-3. Furthermore, stimulation of phospholipase C indicated coupling of A_2B receptors to G_q proteins in HMEC-1. Thus, differential expression of adenosine receptor subtypes contributes to functional heterogeneity of human endothelial cells. A_2B receptors, predominantly expressed in human microvascular cells, modulate expression of angiogenic factors via coupling to G_q, and possibly via G_12/13.