Rats submitted to high altitude (Cerro de Pasco, Perú, 4,340 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%) 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 mitochondrial nitric oxide synthase (mtNOS) activity and expression by >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 -1·day-1) decreased the response of mtNOS to high altitude by 25%. Conversely, NG-nitro-L-arginine methyl ester treatment (8.3 mg·kg-1·day-1) increased such response by 40%, whereas L-arginine treatment (106 mg·kg -1·day-1) had no effect. Nitric oxide (NO) production by mtNOS accounts for ∼49% of total cellular NO production in sea level rats and for ∼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 that is susceptible to be modified by pharmacological treatments. Copyright © 2009 the American Physiological Society.