Although gender difference exists in cardiovascular endothelial function, it remains unclear whether such differences also seen in small intestinal endothelial function. To determine this, male, age-matched proestrus female, castrated male, and 17-estradiol (E2) treated male rats were studied. Dose-response curves to acetylcholine (ACh) and nitroglycerin (NTG) were determined by measuring the changes in perfusion pressure using an isolated small intestinal perfusion model. Endothelium-derived nitric oxide (NO) production/release was indirectly determined by the ability of intact endothelium to suppress serotonin (10^-5 M)-induced perfusion pressure changes. Intestinal tissue levels of NO were also measured. Moreover, plasma levels of androgen and E2 were determined and correlated with ACh (10^-8 M)-induced perfusion pressure reductions. The ACh-induced intestinal perfusion pressure reductions in proestrus females, castrated males, and E2 treated males were significantly higher than in untreated males. NTG-induced perfusion pressure reductions were not significantly different among the groups. The perfusion pressures after the administration of serotonin (10^-5 M) and intestinal tissue levels of NO in proestrus females, castrated-males, and E2-treated males were also significantly higher than in untreated males. Plasma androgen levels in proestrus females, castrated males, and in the E2 treated males were significantly lower compared to untreated males. There was a positive correlation between plasma androgen and ACh-reduced perfusion pressure; however, E2 levels did not show a similar relationship. Thus, androgens appear to play an inhibitory role on small intestinal endothelial function. These properties in male vessels can be modulated by decreasing the level of circulating androgens or by E2 treatment.