Vitamin K was thought to be primarily involved in the production of clotting factors. The recent findings of gamma-carboxyglutamic acid (Gla) - vitamin K-dependent calcium binding amino acid - in various calcified tissues, however, have prompted suggestions that vitamin K may play an important role in calcification mechanism. The present study was designed to investigate the relation between vitamin K and calcification. Morphological, biomechanical and biochemical analyses were undertaken on bones from vitamin K deficient rats raised on vitamin K deficient diet combined with administration of warfarin for three weeks. The Gla-containing proteins were extracted with EDTA from rat or bovine bones, partially isolated with gel filtration, and amino acid analysis was done. Gross roentgenographical and histological abnormalities were not observed in bones from vitamin K deficient rats in which prothrombin level was reduced to about 20 percent. There were no significant differences in values of Young's modulus, which were measured by three-point bending test, between vitamin K deficient and normal bones. No considerable alteration were noted in the protein, calcium or magnesium content of vitamin K deficient bone. Gla content of vitamin K deficient rat bone (per 1,000 residues of glutamic acid) was reduced by approximately 9% compared with that of normal rat bone. This observation supports the hypothesis that Gla biosynthesis in bone may be dependent on vitamin K. Calcium binding property of bovine bone Gla-containing protein was confirmed by the equilibrium dialysis procedure. Moderate amount of hydroxyproline was present in the Gla-containing proteins extracted from rats or bovine bones. It seems possible that biosynthesis of collagen and Gla protein of bone are somehow related, possibly at the level of procollagen. Further investigation is necessary to isolate Gla protein and clarify the correlation between collagen molecule and Gla protein biosynthesis.