Secretory protein I, a protein that is cosecreted with parathormone, has been isolated from bovine parathyroid tissue. The purification procedure was aided by the inclusion in the starting material of fresh tissue that had been incubated with radioactive amino acids to label the newly formed secretory protein I. The isolation of the secretory protein I was then followed by locating the radioactive species. Later, purification was also followed by radioimmunoassay. The procedures included salt fractionation, gel filtration, and two steps of ion-exchange chromatography, yielding a 96-fold purification of secretory protein I. The final product contained two species that were shown to be related by comparison of their tryptic peptides and the release of only a single major residue at each step of the Edman degradation. On the basis of amino acid analysis, secretory protein I contains about 30% acidic amino acid residues, contributing to an isoelectric point of 4.5, and has a minimum molecular weight of about 70000. It contains 2.6% carbohydrate. A radioimmunoassay was established for secretory protein I. A partial amino acid sequence spanning the first 32 residues of the amino-terminal region was obtained. This portion of the structure appeared to be unrelated to those of the known parathyroid hormonal peptides.