Abstract The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [ 3H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla after either intravenous injection of [ 3H]norepinephrine to the rat, or perfusion of the isolated adrenal gland with Krebs-bicarbonate solution containing [ 3H]norepinephrine. The retention of the tritium was inhibited 90% by desipramine. Acute treatment with guanethidine and chronic treatment with 6-hydroxydopamine abolished the secretion of tritium without affecting the secretion of catecholamines evoked at 1 Hz. Nicotine, muscarine and acetylcholine enhanced the secretion of catecholamines but not tritium, whereas tyramine and ephedrine enhanced the secretion of tritium but not catecholamines. It is concluded that chromaffin cells do not possess the norepinephrine uptake mechanism and that the uptake of [ 3H]norepinephrine occurs mainly in sympathetic nerve terminals present in the adrenal gland and the surrounding blood vessels (adrenal and renal veins). The differential localization of [ 3H]norepinephrine and catecholamines allowed us to test the effects of a variety of pharmacological agents that alter neurotransmitter release by acting on receptors on the neuronal membrane, acting on sodium and potassium channels, or acting to alter the intracellular concentrations of adenosine 3′,5′-cyclic monophosphate and protein kinase C. Transmural stimulation (1 Hz for a total of 300 pulses) markedly enhanced the release of catecholamines and tritium which was blocked by tetrodotoxin (sodium channel-blocker) and potentiated by tetraethylammonium and gallamine (potassium channel-blockers). Phentolamine, an alpha adrenergic blocking agent which acts on both alpha-1 and alpha-2 receptors, caused a 3- to 4-fold facilitation of the tritium secretion while inhibiting catecholamine secretion by 45%. [Met]enkephalin almost completely inhibited the evoked-secretion of tritium but had very little effect on the secretion of catecholamines. Forskolin inhibited the tritium secretion by 80% but produced more than a 2-fold facilitation of catecholamine secretion. Phorbol 12,13-dibutyrate caused facilitation of evoked secretion of both catecholamines and tritium. A combination of phorbol ester and forskolin had a synergistic effect on stimulation-evoked secretion of catecholamines, whereas phorbol ester partially reversed the inhibitory effects of forskolin on the tritium secretion. In conclusion, a new technique is described which allows study of the secretion of catecholamines from chromaffin cells and of [ 3H]norepinephrine from sympathetic nerve terminals in one test preparation. Using different test agents, it is demonstrated that chromaffin cells and sympathetic neurons differ considerably in their neurosecretory properties.