Immunoreactive serotonin is demonstrated to be present in 75% of rat adrenal medullary cells using an antibody to serotonin and peroxidase-antiperoxidase immunocytochemical method. The concentration of serotonin in rat adrenals was found to be 7.7 ± 0.1 × 10–6 mol/kg wet weight by high-performance liquid chromatography with electrochemical detection. Drugs that block serotonin synthesis (p-chlorophenylalanine) or deplete biogenic amines (reserpine) diminish immunostaining. The serotonin precursor .L·tryptophan and pargyline, a monoamine oxidase inhibitor, augment staining in reserpine-depleted adrenals. Serotonin is localized in those medullary cells which contain phenylethanolamine-N-methyltransferase, an enzyme which is necessary for the synthesis of epinephrine. We conclude, therefore, that serotonin coexists with epinephrine in rat adrenal medullary chromaffin cells. These results suggest that the adrenal medulla may play a major role in the metabolism of serot