Recent publications shown mitochondrial localization of the enzyme nitric oxide synthase (NOS) in a number of tissues. However, conflicting results about mitochondrial NOS (mtNOS) immunoreactivity and enzymatic activity are available to date in the literature. In this study we purified mitochondria from rat hearts and analysed these preparations for NOS immunoreactivity and activity, showing the presence of either a constitutive (the endothelial isoform) and an inducible NOS immunoreactivity. A basal NOS activity (64.2 +/- 5.1 pmol/mg protein/30 min) was detectable. 1 mM N(G)-Monomethyl-L-arginine (L-NMMA), a competitive inhibitor of all NOS isoforms, caused a drop in NOS activity to 33.8 +/- 1.9 pmol/mg protein/30 min. Simultaneous administration of 10 micro M (S)-2-amino-(1-iminoethylamino)-5- thiopentanoic acid (GW274150), a specific NOS2 inhibitor, together with removal of Ca(2+) and calmodulin (CaM) from the assay buffers, known to interfere with the activity of constitutive NOS isoforms, caused a reduction in NOS activity (17.4 +/- 1.2 pmol/mg protein/30 min). 10 micro M GW274150 reduced NOS activity to 41.6 +/- 4 pmol/mg protein/30 min, while Ca(2+)/CaM withdrawal reduced basal NOS activity to 45.8 +/- 5 pmol/mg protein/30 min. This dual mtNOS machinery is suggested to be involved in modulating cardiac O(2) consumption in different (patho)physiological conditions.