Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-inducedHO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B.-H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375–5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither inducedHO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction ofHO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5′-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or theHO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-inducedHO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation ofHO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-inducedHO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.