Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. Acetylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents (atropine and hexamethonium) and by local anaesthetics (tetracaine), and is dependent upon the concentration of Ca2+ in the incubation medium. In the presence of Ca2+, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca2+ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.