There is separate evidence for peroxynitrite formation and hypoxia-induced cell proliferation in several models of hypoxic pulmonary hypertension. We therefore hypothesized that the stimulation of pulmonary artery smooth muscle cells (PASMCs) proliferation by hypoxia is due to peroxynitrite formation. The effect of hypoxia alone and in combination with ≤ 0.2 μM peroxynitrite on PASMCs was investigated in explants from bovine lungs grown in 1%, 5%, or 10% oxygen for 24 hours with or without peroxynitrite. At 0.1% fetal bovine serum, DNA synthesis of PASMCs (assessed by 3H thymidine incorporation) was increased by transient exposure to 0.2 μM peroxynitrite (by 158% ± 14%, P < 0.01) or to 24 hours of hypoxia (5% oxygen) (by 221% ± 17%, P < 0.01). Results were similar at 2.5% fetal bovine serum. Treatment of PASMCs with 0.2 μM peroxynitrite or 5% O2 hypoxia caused a significant increase in nitrotyrosine formation to a similar extent and intensity. The proliferative response to 0.2 μM peroxynitrite or to the combination of peroxynitrite plus 5% O2 was similar to the effect of 5% O2 alone and was abolished by simultaneous treatment with peroxynitrite scavenger-ebselen (5 μM). Our present data indicate that hypoxia can initiate peroxynitrite-induced proliferative events and suggest a mechanism for the vascular hypertrophy associated with pulmonary hypertension.