Previous studies have demonstrated the uptake of exogenous and storage of endogenous 5-hydroxytryptamine (5-HT) in parenchymal cells of the anterior pituitary. The present experiments were undertaken to test the hypothesis that the endogenous 5-HT of the anterior lobe of the pituitary is costored with beta-luteinizing hormone (beta-LH) within the same secretory granules of gonadotrophs. Electron microscope immunocytochemistry was used to detect 5-HT and beta-LH immunoreactivities in the anterior pituitary glands of mice and bats. Primary antisera generated in different species of animals to these two antigens were localized with appropriate species-specific secondary antisera coupled to colloidal gold particles of different sizes. This enabled 5-HT and beta-LH immunoreactivities to be demonstrated simultaneously on ultrathin sections of fixed anterior lobe tissue mounted on electron microscope (EM) grids. In both bats and mice 5-HT immunoreactivity, identified by immunostaining of beta-LH, was found in gonadotrophs, and in no other cell type. Within gonadotrophs about 25% of the secretory granules were labeled by antisera to both 5-HT and beta-LH, although 100% of granules reacted with the antiserum to B-LH. No secretory granules were found that were immunostained only by the antiserum to 5-HT. It is concluded that endogenous 5-HT may be a normal constituent of mammalian gonadotrophs and that it is colocalized with beta-LH in at least a subset of the secretory granules of these cells. It cannot yet be concluded that gonadotrophs synthesize 5-HT as well as taking it up from the ambient medium.(ABSTRACT TRUNCATED AT 250 WORDS)