The gamma-carboxyglutamate-containing protein from human bone has been isolated from proteins released upon demineralization of bone by using a combination of gel filtration and immunoadsorption onto a column of immobilized antibody. The sequence of the protein has been determined by automatic sequence analysis of the whole protein and of peptides isolated from tryptic and Staphylococcus aureus protease digests. The protein consists of 49 amino acids in a single polypeptide chain, contains 2 residues of gamma-carboxyglutamic acid, and a single disulfide bond. There are 4 translational substitutions between the human and calf bone proteins and 23 translational substitutions between the human and swordfish bone proteins. Glu-17 was found to be only 9% gamma-carboxylated, while sequence positions 21 and 24 are fully gamma-carboxylated. The identification of glutamic acid at position 17 represents the first instance where a partially gamma-carboxylated glutamate has been found in a sequence position which is to the NH2-terminal side of a gamma-carboxyglutamate residue. The possible significance of this observation is discussed.