Nitrovasodilators, such as sodium nitroprusside (SNP), release nitric oxide (NO) and stimulate intestinal electrolyte transport. However, the second messengers involved in this process are unknown. NO stimulates soluble guanylate cyclase activity in other tissues, but stimulation of this enzyme has not previously been described for intestine. We report a 20-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) production by radioimmunoassay in colonic mucosal strips stimulated with SNP. SNP also caused a significant increase in prostaglandin (PG) E2 release but did not stimulate release of the prostanoids thromboxane B2 or 6-keto-PGF1alpha. Stimulation of isolated colonic crypts and the remaining subepithelial mucosa demonstrated that the latter was the major source of the increases in cGMP and PGE2. Immunostaining of colonic mucosa revealed minimal basal cGMP immunoreactivity but large increases in abundance, localizing to the subepithelium, after SNP treatment. Under basal conditions, there was diffuse immunostaining for constitutive NO synthase in both the epithelial and subepithelial compartments, which was corroborated with NADPH diaphorase staining. In conclusion, SNP was an NO donor stimulates production of cGMP and PGE2 from the subepithelium. NO may be an important mediator of colonic secretion and other processes predominantly via its direct effects on cells of the lamina propria.