Chromogranin A (CGA) is a ubiquitous 48-kDa secretory protein present in adrenal medulla, anterior pituitary, central and peripheral nervous system, endocrine gut, thyroid, parathyroid, and endocrine pancreas. Recently, we have demonstrated that the protein could be a precursor of bioactive peptides capable of modulating catecholamine secretion from cultured adrenal medullary chromaffin cells. Here we cleaved CGA purified from bovine chromaffin granules with endoproteinase Lys-C, and we isolated and partially sequenced the peptide inhibiting catecholamine secretion from cultured chromaffin cells. A corresponding synthetic peptide composed of the first 20 N-terminal amino acids produced a dose-dependent inhibition in the 10(-9) to 10(-6) M range (with an ID50 of 5 nM) of the catecholamine secretion evoked by carbamoylcholine or by potassium at a depolarizing concentration. This peptide affected secretagogue-induced calcium fluxes but did not alter sodium fluxes. It was found to increase desensitization of cell responses and to modify the kinetics of catecholamine release. Our results indicate that the peptide is extracellularly generated from CGA by a calcium-dependent proteolytic mechanism. We suggest that this peptide, named chromostatin, may be an endocrine modulator of catecholamine-associated responses.