Chromogranin A (CgA) is a glycoprotein present in secretory granules of endocrine cells. In the parathyroid, it is costored and cosecreted with parathormone (PTH) in response to hypocalcemia. CgA is the precursor of several bioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA(347-419)) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca(2+)-stimulated parathyroid secretion. Full activity resides in its first 19 residues. In order to determine if PARA or PARA-derived peptides are natural products of the parathyroid, we generated an antiserum directed against pCgA(347-359) corresponding to the bioactive N-terminal sequence of pPARA (pPARA(1-13) antiserum), and developed a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPARA(1-13) immunoactivity in extracts and secretion medium of porcine parathyroid glands. Continuous and pulse-chase radiolabeling studies, along with immunoprecipitation using PARA(1-13) antiserum demonstrate that a newly-synthesized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradation of the 11 kDa PARA-related peptide band by Edman degradation yielded three major N-terminal sequences: S-K-M-D-R-L-A-K-E-L-(residues 313-322), D-R-L-A-K-E-L-T-A-E-(residues 316-325), and A-K-E-L-T-A-E-K-R-L-(residues 319-329), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indicate that the C-termini of these peptides extended variably to residues 384-401 of pCgA. These results demonstrate that processing of CgA by the parathyroid gland generates bioactive PARA-related peptides that could affect the gland's secretory activity.