Rats submitted to high altitude (Cerro de Pasco, Perú, 4340 m, PO₂=12.2 kPa) for up to 84 days showed a physiological adaptive response with decreased body weight gain (15 %), increased right ventricle weight (100 %) and increased hematocrit (40 %), as compared with sea level animals. These classical parameters of adaptation to high altitude were accompanied by an increase in heart mitochondrial enzymes: complexes I-III activity by 34 % and mtNOS activity and expression by more than 75 %. The hyperbolic increase for mtNOS activity during adaptation to high altitude was similar to the observed pattern for hematocrit. Hematocrit and mtNOS activity mean values correlated linearly (R² = 0.75, P 0.05). Chronic treatment for 28 days with sildenafil (50 mg/kg.day) decreased the response of mtNOS to high altitude by 25 %. Conversely, L-NAME treatment (8.3 mg/kg.day) increased such response by 40 %, whereas L-arginine treatment (106 mg/kg.day) had no effect. Nitric oxide production by mtNOS accounts for about 49 % of total cellular NO production in sea level rats and for about 54 % in rats exposed to high altitude for 84 days. It is concluded that mtNOS is a substantial source of cardiac NO, a factor in the adaptive response to sustained heart hypoxia which is susceptible to be modified by pharmacological treatments.