The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.