We report the identification and the primary structure of a novel neuroendocrine peptide which inhibits aldosterone secretion. The peptide was isolated from 1.5 x 10(10) cultured bovine chromaffin cells by reversed phase and ion exchange HPLC. It is chromatographically and structurally distinct from atrial natriuretic factor (ANF), which we formely identified in chromaffin tissue. This new Aldosterone Secretion Inhibitory Factor (ASIF) is predominant in cultured chromaffin cells and cross-reacts with ANF receptor sites involved in the inhibition of aldosterone production in zona glomerulosa cells. The sequence of this 35-amino acid peptide includes the shorter fragment isolated from porcine brain confirming its neuropeptide character. ASIF might be involved in concert with ANF in the paracrine regulation of aldosterone secretion and the presence of both peptides in cultured chromaffin cells indicates that this system can be used as a model for studying natriuretic neuropeptide production.