We investigated adenosine stimulation of DNA synthesis in human endothelial cells by measuring [3H]thymidine incorporation in cultures derived from human umbilical veins. After 18 h of exposure to adenosine in serum-free medium, endothelial cell [3H]thymidine incorporation was increased by 30-64%. Adenosine-induced DNA synthesis was not mimicked by adenosine receptor agonists and was not inhibited by adenosine receptor antagonists. Adenosine-induced DNA synthesis was inhibited 81% by 100 microM 5'-(N,N-dimethyl)amiloride, an inhibitor of Na+/H+ exchange, and was totally inhibited by 10 microM 2',4'-dibromoacetophenone, an inhibitor of phospholipase A2 (PLA2). Adenosine increased adenosine 3',5'-cyclic monophosphate levels in endothelial cells, but adenosine-induced DNA synthesis was not inhibited by the protein kinase A (PKA) inhibitor Rp-cAMPS. Both ATP and the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) increased DNA synthesis in human endothelial cells. Stimulation by ATP was inhibited by the P2-receptor antagonist suramin, and PMA stimulation was inhibited by the protein kinase C (PKC) inhibitor H-7. Neither suramin nor H-7 inhibited adenosine-stimulated DNA synthesis. The results suggest that Na+/H+ exchange and PLA2 are involved in adenosine-induced DNA synthesis in cultures of human endothelial cells independently of adenosine receptor, PKA, or PKC activation.