Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H, 10% O₂) with or without heparin (1200 U/kg/d) and/or the NO synthase (NOS) inhibitor N^G-nitro-L-arginine (L-NAME, 20 mg/kg/d) for 3 d or 3 wk. Heparin attenuated increases in pulmonary artery pressure, the percent of muscular pulmonary vessels and their medial thickness induced by 3 weeks of H. Importantly, while L-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and 3'5'cyclic guanosine monophosphate (cGMP) levels at 3 d and 3 wks, and endothelial NOS protein expression at 3 d but not at 3 wk. In vitro, heparin (10 and 100 U/kg/ml) increased cGMP levels after 10 min and 24 hr in N and anoxic (0% O₂) endothelial cell/smooth muscle cell (SMC) coculture. SMC proliferation, assessed by BrdU-incorporation during a 3 hr incubation period, was decreased by heparin under N, but not anoxic condition. The anti-proliferative effects of heparin were not altered by L-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO-mediated. Heparin increases cGMP in vitro, however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO-independent.