The possible role of serotonin (5-hydroxytryptamine, 5-HT) in the secretory activity of the subcommissural organ (SCO) was investigated in the cat by using 5-HT immunohistochemistry and Gomori's secretory staining technique. Administration of para-chlorophenylalanine, a potent inhibitor of 5-HT synthesis, was found to produce a marked decrease in both 5-HT-immunoreactive materials and secretory granules in the SCO. In contrast, administration of 5-hydroxytryptophan, an immediate precursor of 5-HT, to animals pretreated with para-chlorophenylalanine resulted in an increase in the amount of both secretory granules and 5-HT immunoreactivities in this circumventricular organ. These results strongly suggest that the secretory activity of the SCO of cats is regulated by 5-HT.