Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous NOS inhibitors, their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured nitric oxide synthase (NOS) and arginase activities, and NOS protein expression. Twenty four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cyclic GMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group. Whereas, the arginase, which shares L-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cyclic GMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the down-regulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the down-regulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.