In addition to the three known forms of nitric oxide synthase (NOS) in the heart, it has recently been proposed that NOS is also present in mitochondria. However, studies are controversial due to the possibility of contamination by non-mitochondrial NOS, and because none of the known forms of NOS contain a mitochondrial targeting sequence. We investigated whether NOS was present in isolated mitochondria using antibodies against all three forms of NOS (endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS)). Crude fractions of heart and liver mitochondria were obtained by differential centrifugation, and 35% Percoll was used to obtain highly purified mitochondria, as tested using antibodies against subcellular marker proteins: cyclophilin D, mitochondrial marker; monocarboxylate transporter-1, plasma membrane marker; ryanodine receptor, sarcoplasmic reticulum marker (heart mitochondria only) and catalase, peroxisomal marker (liver only). Western blotting using antibodies against eNOS and iNOS revealed that these isoforms were not present in either heart or liver purified mitochondria (whereas whole heart or liver lysate tested positive). We used five different antibodies against nNOS, and again failed to detect anything in purified heart mitochondria. However, in purified liver mitochondria one of the nNOS antibodies revealed the presence of a band at the correct molecular weight. We are currently determining whether this is indeed nNOS. In addition, we will assay for NOS activity in the purified mitochondria. Nitric oxide can inhibit mitochondrial respiration, so the existence of mitochondrial NOS may provide an important modulatory mechanism for respiration under either physiological or pathological conditions.