Osteocalcin was purified in high yield and to homogeneity from the diaphysis of dog femora by the following steps: (1) acid demineralization of bone powder, (2) solid-phase extraction of acid-soluble proteins on Sep-Pak C18 cartridges, (3) gel filtration on Sephadex G-50, and (4) fast protein liquid chromatography on an Accell-QMA anion-exchange column. Starting from 30 g washed bone powder, approximately 7-10 mg pure protein was obtained in 2 days. The key step is the initial solid-phase extraction of osteocalcin from a large volume of a demineralized bone solution. The primary structure was established by automated sequence analyses of two tryptic peptides, of two endoproteinase Glu-C carboxy-terminal peptides, and of the first 30 amino acid residues of the intact protein. Dog osteocalcin contains 49 amino acids, has a molecular mass of 5654 daltons, contains no Thr, Met, Hyp, or Trp, has a disulfide bond between Cys 23 and 29, and is fully gamma-carboxylated at residues 17, 21, and 24. Dog osteocalcin does not contain a pair of basic amino acids found at positions 43-44 in most other osteocalcins from mammals and birds. A computer search for homology indicated 88, 90, 84, 88, 66, and 57% sequence identity of dog osteocalcin with human, bovine, cat, monkey, chicken, and swordfish osteocalcin, respectively, and weaker homologies with the gamma-carboxylated domains of blood-clotting proteins and the Pro-rich N-terminal extensions of myosin light-chain A1 and beta-crystalline B1. The possible relevance of these homologies to the structure and potential functions of osteocalcin is discussed.