The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small size pulmonary arteries, and in alveolar walls of hypoxic lungs. 12-LO protein expression is increased in hypoxic cultured rat pulmonary artery SMCs. 12-HETE at concentrations as low as 10^-5 µM stimulated proliferation of pulmonary artery SMCs. 12-HETE induced ERK 1/ERK 2 phosphorylation, but had no effect on p38 kinase expression as assessed by Western blotting. 12-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor, PD98059, but not by the p38 MAPK inhibitor, SB202190. Hypoxia (3%) - stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.