The novel chromogranin A fragment catestatin (bovine chromogranin A_344−364; RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC₅₀, ∼0.2–0.3μ m) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A_344−358. Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met³⁴⁶, Leu³⁴⁸, Phe³⁵⁰, Arg³⁵¹, Arg³⁵³, Gly³⁵⁴, Tyr³⁵⁵, Phe³⁵⁷, and Arg³⁵⁸ on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg³⁴⁴, Met³⁴⁶, Leu³⁴⁸, Ser³⁴⁹, Phe³⁵⁰, Arg³⁵³, Gly³⁵⁴, Tyr³⁵⁵, Gly³⁵⁶, and Arg³⁵⁸. We conclude that a small, 15-amino acid core of catestatin (chromogranin A_344−358) is sufficient to exert the peptide’s typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.