Galanin (Gal), a 29-amino acid C-terminally amidated neuropeptide, is widely distributed throughout the central and peripheral nervous system. The primary structures of rat and bovine Gals were derived from the cDNA sequences of their precursors. To elucidate the structure of human Gal (hGal), we extracted 280 postmortem pituitaries in trifluoroacetic acid and purified hGal binding activity, by three successive HPLC steps, to homogeneity based on a radioreceptor assay. The primary structure of hGal was determined by automatic Edman degradation to be Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu- Gly-Pro-His-Ala-Val-Gly-Asn-His-Arg-Ser-Phe-Ser-Asp-Lys-Asn-Gly-Leu-Thr- Ser-COOH. The structure was confirmed by plasma desorption time-of-flight mass spectrometry, revealing a mass of 3156.1. Compared to the 29-residue porcine, rat, and bovine Gals, hGal uniquely comprises 30 amino acids possessing an additional nonamidated serine residue as C terminus. The nonamidated carboxylic group at the C terminus was proven by synthesis of amidated and nonamidated hGal and by mass spectrometry after selective methylation of all free carboxylic groups. Synthetic hGal possesses full biological activity on isolated rat fundus muscle strips.