Nucleotides such as ATP and UTP are emerging as a ubiquitous family of extracellular signaling molecules. Nucleotides are released from cells by a variety of pathophysiologic stimuli and have been shown to play a key role in transducing mitogenic, contractile, metabolic, and secretory signals in a variety of cells through release and subsequent binding to the P2 (P2X and P2Y) family of purinergic receptors. However, little is known regarding the role of ATP in hypoxia-induced vascular cell responses. In a neonatal model of hypoxic pulmonary hypertension, we showed that Pulmonary Artery (PA) adventitial fibroblasts proliferate both in vivo and in vitro in response to hypoxic conditions. We therefore examined the hypothesis that hypoxia-induced adventitial fibroblast proliferation would be mediated by hypoxia-induced changes in ATP release and/or its extracellular degradation. We found that acute hypoxia (3%O2 10-60 min) increased extracellular ATP concentrations in adventitial fibroblasts and that chronic hypoxia (3%O2, 14-30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ectonucleotidase(s) suggesting that at least two different cellular mechanisms may contribute to elevated extracellular ATP levels. Exogenous ATP (100 μM) stimulated thymidine incorporation and increased the phosphorylation of Akt, Erk1/2, mTOR, and p70S6K in fibroblasts as did UTP, UDP, ADP, ADPβS, MeSATP, αβMeATP, BzATP and some other agonists, indicating that both P2Y and P2X purinoceptors mediate mitogenic responses. PCR analysis revealed that adventitial fibroblasts express P2Y1,2,6 as well as P2X2,4,6,7 receptor subtypes. The rank order of potency of various agonists to activate each individual kinase pathway (ERK1/2, PI3K/Akt or mTOR/p70S6K) indicates that in these cells, purinergic receptors are coupled to the proliferative responses in a pathway-specific manner. Importantly, in this ATP-activated signaling network, a translational pathway, involving mTOR, p70S6K and S6 ribosomal protein, plays a central role in integrating Erk1/2 and PI3K/Akt pathways. We also found that ATP (100μM) and hypoxia (3%O2), induced expression and activation of the Egr-1 transcription factor, and both stimuli acted, in part, through Gαi-initiated ERK1/2 signaling pathway. Apyrase (2.5U/ml), as well as the non-selective P2 receptor antagonists, suramin, cibacron blue 3GA, and PPADS (all used at 100μM) attenuated hypoxia- and ATP-induced DNA synthesis, indicating an activation and a functional role of P2Y/P2X purinoceptors in hypoxia-induced proliferative responses. In addition, suramin, cibacron blue 3GA and apyrase, markedly attenuated hypoxia-induced ERK1/2 activation and Egr-1 expression. Collectively, our findings demonstrate that PA adventitial fibroblasts can be considered as endogenous source and a target of extracellular nucleotides within the vascular wall and that a hypoxia-induced autocrine loop of ATP signaling plays a critical role in the regulation of fibroblast proliferation under hypoxic conditions.