Serotonin [5-hydroxytryptamine (5-HT)] is a constituent of the mammalian pituitary gland. We have used light and electron microscopic immunocytochemistry to locate immunoreactivity in the pituitary glands of mice and bats. In addition, we have compared the distribution of endogenous 5-HT immunoreactivity with that of exogenous [3H]5-HT taken up by elements of the pituitaries and detected by radioautography. 5-HT immunoreactivity was found in neurites in the posterior and intermediate lobes of the glands. These neurites also took up [3H]5-HT. The neural elements reactive with antisera to 5-HT or which labeled with [3H]5-HT differed in their distribution from neurites that labeled with [3H]dopamine or [3H]norepinephrine; moreover, lesioning of catecholaminergic neurites with 6-hydroxydopamine had no effect on the serotonergic structures. 5-HT immunoreactivity was also found in endocrine cells of the anterior lobes of the pituitaries of both species. No 5-HT was detected in neural elements of the anterior lobe, and in fact, no staining with nerve-specific antibodies to neurofilament proteins was found in the anterior pituitary. The ultrastructure of the granules of the 5-HT-immunoreactive endocrine cells suggested that they might be a subset of gonadotrophs, the cell type previously shown to take up exogenous [3H] 5-HT. 5-HT immunoreactivity was found within the granules of these cells. In bats, 5-HT immunoreactivity was present in the anterior pituitaries of active animals when 5-HT levels were high, but was greatly diminished in glands of hibernating animals when the 5-HT content was low. It is concluded that some cells that may be gonadotrophs contain endogenous 5-HT and also have an uptake mechanism for the amine. The function of 5-HT in the anterior pituitary remains to be determined, but 5-HT has previously been shown to inhibit the secretagogue action of LHRH on gonadotrophs.