Previous studies raised the possibility that nitric oxide synthase is present in heart mitochondria (mtNOS) and the existence of such an enzyme became generally accepted. However, original experimental evidence is rather scarce and positive identification of the enzyme is lacking. We aimed to detect an NOS protein in human and mouse heart mitochondria and to measure the level of NO released from the organelles. Western blotting with 7 different anti-NOS antibodies failed to detect a NOS-like protein in mitochondria. Immunoprecipitation or substrate-affinity purification of the samples concentrated NOS in control preparations but not in mitochondria. Release of NO from live respiring human mitochondria was below 2 ppb after 45 min of incubation. In a bioassay system, mitochondrial suspension failed to cause vasodilation of human mammary artery segments. These results indicate that mitochondria do not produce physiologically relevant quantities of NO in the heart and are unlikely to have any physiological importance as NO donors, nor do they contain a recognizable mtNOS enzyme.