Activation of the alpha-smooth muscle actin (alpha-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates alpha-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased alpha-SMA expression is controlled at the transcriptional level because the alpha-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the alpha-SMA gene was mimicked by overexpression of constitutively active Galphai2 (alphai2Q205L) but not Galpha16 (alpha-16Q212L). Blockade of hypoxia-induced alpha-SMA expression with pertussis toxin, a Galphai antagonist, confirmed a role for Galphai in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated alpha-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and alpha-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-beta1 blocked only 30% of the hypoxia-induced alpha-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of alpha-SMA, and this increase in alpha-SMA level occurs through Galphai- and JNK-dependent signaling pathways.