Development of a sensitive radioimmunoassay for the vitamin K-dependent bone protein osteocalcin in avian species has provided new information on the biosynthesis of this protein in bone. Chicken osteocalcin shares many structural features, including the sequence positions of its 3 gamma-carboxyglutamic acid (Gla) residues, with osteocalcins of human, monkey, cow, and rat, but is cryptic in the radioimmunoassays for these species. In the chicken assay system, the intact 50-residue (Mr = 5670) protein is required for immunoreactivity. Reduction and alkylation of the disulfide bond (Cys 23-Cys 29) or tryptic removal of the COOH-terminal pentapeptide abolish antibody binding activity. Decarboxylation of the 3 Gla residues enhances the affinity for antibody by 1.5- to 2-fold. Osteocalcin appears coincident with the very earliest detectable perichondral mineralization in developing long bone (tibiotarsus) of the 7- to 8-day-old chick embryo (stages 31-33). However, amino acid analysis demonstrates an excess of Gla in embryonic bone compared to the level of osteocalcin by radioimmunoassay. Two independent experimental approaches have partially resolved this paradox. First, extraction and gel filtration in 4 M guanidine hydrochloride of total bone proteins has revealed high molecular weight species which share antigenic determinants with osteocalcin, namely, 10,000 (+/- 1,000), 15,000 (+/- 2,000), 35,000 (+/- 5,000), and 85,000 (+/- 15,000), in addition to 5,670 osteocalcin. Second, a selective 3H exchange labeling procedure for Gla residues has revealed Gla-containing proteins in bone in the same molecular weight classes. One or more of these may represent precursors in the biosynthetic pathway for osteocalcin.