This study investigated the release of nitric oxide (NO) from glyceryl trinitrate (GTN) and SIN-1 in Langendorff rabbit hearts. Infusion of either GTN (10–40µ M) or SIN-1 (0.45–4.5 µ M) into the coronary inflow tract resulted in a decrease in coronary perfusion pressure and NO release (oxyhemoglobin technique) into the coronary effluent. NO release fom SIN-1 occurred spontaneously whereas passage through the coronary circulation, i.e. active metabolism, was required for NO release from GTN. Removal of the coronary endothelium and blockade of endothelial NO formation did not affect NO release from GTN and SIN-1. In GTN-tolerant hearts, there was a considerable inhibition of GTN- but not SIN-1-induced NO formation and coronary vasodilation. These data suggest (1) that metabolic NO release from GTN occurs during passage of the coronary circulation and is independent of the presence of endothelium, and (2) reduced NO release is a major cause of nitrate tolerance.