Nitroglycerin (NTG), endothelium-derived relaxing factor (EDRF) and atrial natriuretic peptide (ANP) dilate vessels by a final common pathway (cyclic guanosine monophosphate-dependent mechanism). Thus, these vasodilators may potentially influence the vasodilator properties of each other. We examined whether tolerance to NTG influences vasorelaxation to ANP, EDRF and other nitrovasodilators, isosorbide dinitrate (ISDN) and sodium nitroprusside (SNP). Concentration-response curves for various vasodilators during stable contraction to PGF<sub>2α</sub> were examined before and after the induction of NTG tolerance. Rings exposed to NTG (100 µ M) for 60 min showed marked impairment of vasorelaxation to NTG itself. This NTG tolerance significantly attenuated vasorelaxation to either ISDN, SNP or EDRF stimulated by bradykinin. In contrast, the NTG tolerance did not alter vasorelaxation to either ANP or isoproterenol. The attenuated responses to NTG, ISDN, SNP and bradykinin recovered in a time-dependent manner during the subsequent washout period, suggesting this inhibition is reversible. The denudation of the endothelium significantly sensitized relaxation to NTG but did not change relaxation to ANP. These results suggest that (1) vasorelaxations to nitrovasodilators and EDRF are significantly attenuated by NTG tolerance and (2) ANP-induced vasorelaxation is not affected by exposure to either exogenous nitrovasodilator or endogenous endothelium-derived nitric oxide. ANP may be a potentially beneficial vasodilator in arteries which have developed tolerance to nitrovasodilators.