The role of intracellular guanosine 3',5'-cyclic monophosphate concentration ([cGMP]_i) in nitric oxide (NO)-mediated relaxations in the uterus has become controversial. We found the NO donor S-nitroso-L-cysteine (CysNO) to potently (IC₅₀ = 30 nM) inhibit spontaneous contractions in the nonpregnant human myometrium. CysNO treatment increased [cGMP]_i significantly (P < 0.001), and this increase was blocked by the guanylyl cyclase inhibitors methylene blue (10 µM) or LY-83583 (1 µM); however, pretreatment with these guanylyl cyclase inhibitors failed to block CysNO-mediated relaxations. Intracellular cAMP concentrations were not altered by treatment of tissues with 10 µM CysNO. Incubation with the cGMP analogs 8-bromo-cGMP or -phenyl-1,N²-etheno-cGMP did not significantly affect spontaneous contractility. Pretreatment of tissues with charybdotoxin [a calcium-dependent potassium channel (BK) blocker] completely reversed CysNO-induced relaxations. We conclude that NO is a potent inhibitor of spontaneous contractile activity in the nonpregnant human uterus and that, although guanylyl cyclase and BK activities are increased by NO, increases in [cGMP]_i are not required for NO-induced relaxations in this tissue.