Serotonin release from rabbit enterochromaffin cells located in the mucosal epithelium of the small intestine was studied in vitro. Serotonin release from both the serosal and mucosal sides of the small intestine was measured. The addition of muscarinic but not nicotinic cholinergic agonists to the serosal medium resulted in a large but transient increase in serotonin release from the serosal but not the mucosal side of the intestine. Mucosal addition of these agents was ineffective. Serotonin release stimulated by the cholinergic agonist carbachol appeared to be dependent upon influx of extracellular Ca++ for the following reasons: 1) depletion of serosal Ca++ inhibited carbachol-stimulated release; 2) carbachol-stimulated serotonin release was blocked by the inorganic calcium channel blockers Co++, Ni++, Cd++, La and Gd; and 3) serosal serotonin release was increased by the Ca++ ionophore, ionomycin, and by Ba++. The addition of 8-bromoadenosine cyclic AMP or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, to the serosal medium produced a sustained elevation of serosal serotonin release. 8-bromoadenosine-cyclic AMP-stimulated release was not blocked by depleting extracellular Ca++. Forskolin, a compound which stimulates adenylate cyclase, also stimulated serosal serotonin release. 8-bromoadenosine-cGMP had no effect on serotonin release. Somatostatin (10(-8)-10(-6) M) caused a dose-dependent inhibition of carbachol-stimulated serotonin release. Somatostatin (10(-6) M) only partially inhibited serotonin release stimulated by 8-bromoadenosine-cyclic AMP, 3-isobutyl-1-methylxanthine and forskolin and had no effect on release stimulated by Ba++. The results suggest potential roles for both calcium and cyclic nucleotides in the regulation of serotonin release.