We and others have provided indirect evidence for the presence of a constitutive nitric oxide synthase (cNOS) in the mammalian heart. We now provide more direct evidence for the regulation of a myocardial cNOS in the hearts of patients undergoing elective cardiopulmonary bypass (CPB). cNOS enzyme activity was demonstrable in both cytosolic (8.3 ± 0.02 pmol/min/mg) and membrane (11.1 ± 0.4 pmol/min/mg) preparations derived from human atrial pectinate muscles obtained at the rime of CPB (n = 6). Plasma nitrite (NO2-) + plasma nitrate (NO3-) levels from the beating hearts of patients before bypass were reduced from 146 ± 33 to 5.1 ± 50 pmol/min/g after cardiac arrest during CPB (n = 23; p < 0.002 by Student's t test). Thus, the human myocardium constitutively produces nitric oxide that is regulated by the contractile state of the heart.