Important autocrine/paracrine functions for the adenine nucleotides have been proposed in several tissues. We addressed the possibility that extracellular ATP would modulate/mediate hypoxia-induced adventitial fibroblast growth. Acute hypoxia (3% O2, 10-60 min) increased extracellular ATP concentrations in adventitial fibroblasts and in lung microvascular endothelial cells, and chronic hypoxia (3% O2, 14-30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ecto-nucleotidase(s). Exogenous ATP stimulated [3H]thymidine incorporation in fibroblasts as did UTP, ADPβ, 2-methylthioadenosine triphosphate, adenosine 5′-(α,β-methylene)triphosphate, and benzoylbenzoyl-ATP (2′-3′-O-(4-benzoylbenzoyl)-ATP), indicating that both P2Y and P2X purinoceptors can mediate mitogenic responses. Suramin (100 μM), Cibacron blue 3GA (100 μM), and pyridoxalphosphate-6-azophenyl-2′,-4′-disulfonic acid (100 μM) as well as apyrase (5 units/ml) attenuated hypoxia- and ATP-induced and DNA synthesis, indicating activation and a functional role of purinoceptors under hypoxic conditions. ATP-induced DNA synthesis was augmented by hypoxia in an additive fashion, whereas ATP and hypoxia synergistically increased growth factor-induced DNA synthesis, again suggesting that ATP and hypoxia utilize similar signaling pathways to induce proliferation. Indeed, we found that ATP (100 μM) and hypoxia (3% O2) induced expression and activation of Egr-1 transcription factor, and both stimuli acted, in part, through a Gαi/ERK1/2-dependent signaling pathway. Suramin, Cibacron blue 3GA, and apyrase attenuated hypoxia-induced ERK1/2 activation and Egr-1 expression. We conclude that hypoxia induces ATP release from endothelial cells and fibroblasts and that the activation of P2 purinoceptors is involved in the regulation of DNA synthesis by fibroblasts under hypoxic conditions.