We previously found serotonin in the adrenal medulla of the rat using immunocytochemical methods. Serotonin immunoreactivity was found in 75% of the medullary cells and by double staining techniques was found to be present in epinephrine-containing cells. In order to better understand the chemical and pharmacological nature of the adrenomedullary serotonin containing cells, we undertook the present studies to characterize the cells' response to a number of agents which have been used to assess biochemical relationships in other serotonin systems. The responsiveness of the serotonin-containing cells to these agents was determined by immunocytochemical methods directed against serotonin. Reserpine, a monoamine depleting agent, caused a significant reduction in the amount of serotonin immunostaining. Parachloroamphetamine (PCA), a specific serotonin releaser, administered in vivo had a minimal effect on the depletion of serotonin immunoreactivity. However, when applied in vitro, PCA always caused a dose dependent depletion; the effect of PCA in vitro was blocked by pretreatment and co-incubation with serotonin-uptake inhibitor, fluoxitine. Exposure to parachlorophenylalanine (PCPA) which inhibits serotonin synthesis, resulted in a marked reduction of immunostaining of most cells. The small population of cells which still stained intensely after PCPA treatment was depleted by incubation with the specific releasing agent, parachloroamphetamine (PCA) in vitro. Restoration of control-like immunostaining after reserpine-depletion occurred with treatment with the serotonin precursor, L-tryptophan, both in vivo and in vitro. From these results we infer the existence and regulation of adrenomedullary serotonin which is similar but not identical to that reported for serotonin neurons of the CNS.