It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3 morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.