Nitrovasodilators relax vascular smooth muscle by stimulating soluble guanylate cyclase (GC). The resulting rise in cGMP probably initiates Ca extrusion from the smooth muscle cell which causes relaxation. Since repeated administration of organic nitrates, particularly nitroglycerin, leads to tolerance, i.e. a decrease in the vasodilator effect, it was studied whether (a) tolerance was a peripheral phenomenon occurring in the vascular smooth muscle, and (b) was due to an impairment of GC activation. In isolated circular strips of bovine coronary arteries, 90 min pretreatment with nitroglycerin greatly lowered the relaxing as well as the cGMP increasing response to nitroglycerin, indicating tolerance induction. Tolerance, although to a lesser extent, was also obtained with other organic nitrates under similar conditions, including IS 5-MN. Little (nitroprusside Na) to negligible tolerance was obtained with sodium nitrate and SIN-1, the active metabolite of molsidomine. The latter group of drugs stimulated soluble GC in vitro in the absence of cysteine whereas organic nitrates required the presence of this thiol. Preincubation with nitroglycerin almost completely inactivated GC whereas other organic nitrates had little effect. The results indicate that tolerance is caused by an impairment of GC function in the smooth muscle cell, particularly when elicited by nitroglycerin, and that differences in the degree of tolerance development by various nitrovasodilators are possibly due to different mechanisms of activation and inactivation of GC as well as differences in cysteine requirement.