We investigated the role of K⁺ channels and intracellular Ca²⁺ stores in the relaxations induced by the NO donor 3-morpholinosydnonimine (SIN-1) and 8-bromo-cGMP (8-BrcGMP), 8-(4-chlorophenylthio)-cGMP (pCPT-cGMP), and ,-methylene-ATP in isolated segments of rat ileum. The inhibitory responses to SIN-1 and the cGMP analogs were not influenced by the K⁺ blockers apamin, charybdotoxin, iberiotoxin, or glibenclamide, whereas relaxations induced by ,-methylene-ATP were abolished by apamin and tetraethylammonium. The NO-donor SIN-1 and the cGMP analogs were able to inhibit contractions induced by activation of L-type Ca²⁺ channels (BAY-K-8644), by carbachol (CCh), and by cyclopiazonic acid (CPA), a blocker of sarcoplasmic Ca²⁺-ATPase. However, the inhibition of the combined CPA and CCh response was reduced and the dose-response curve of SIN-1 shifted to the right. Intracellular Ca²⁺ stores were emptied by incubation in Ca²⁺-free buffer and repetitive stimulation with CCh or BAY-K-8644. After restoration of extracellular Ca²⁺, the inhibitory effect of SIN-1 and pCPT-cGMP was only attenuated, whereas in the additional presence of CPA, the inhibitory effect of SIN-1 was blocked and the effect of 8-BrcGMP reduced. Thus depleting intracellular Ca²⁺ stores attenuated the effect of SIN-1 and 8-BrcGMP, suggesting an involvement of functional Ca²⁺ stores.