Synthesis of osteocalcin, a protein of the extracellular matrix of bone, is reported to be stimulated by supraphysiological concentrations of vitamin D. Although its role remains unknown, osteocalcin has been hypothesized by Price and coworkers to retard bone mineralization. The objective of the proposed project is to learn if osteocalcin plays a role in inhibition of mineralization by vitamin D in cultures of isolated murine osteoblasts. The hypothesis to be tested is that supraphysiological concentrations of vitamin D inhibit osteoblast mineralization in culture by stimulating osteocalcin synthesis. The specific aims of the project are to set up a radioimmunoassay (RIA) for osteocalcin; to measure osteocalcin synthesis by osteoblasts cultured in the absence and presence of vitamin D to compare osteocalcin synthesis in sparse cultures sensitive to inhibition by vitamin D with that in dense cultures refractory to inhibition by vitamin D; to determine if compounds (e.g., parathyroid hormone, glucocorticoids) reported to decrease vitamin D-induced osteocalcin synthesis, also alter mineralization; and to use warfarin to block osteocalcin synthesis and establish if vitamin D still can inhibit mineralization by cultured cells. Isolated osteoblasts from neonatal calvariae of mice will be cultured over a 3-week period in collagen gels and osteocalcin in gels and culture medium assayed by RIA. Other cellular parameters which will be routinely assayed are cell number, alkaline phosphatase activity, and mineralization. Data will be expressed per 10 6 cells. The results of these studies will indicate if supraphysiological concentrations of vitamin D inhibit mineralization by stimulating osteocalcin synthesis. They also will contribute to understanding biochemical regulation of normal bone mineralization as well as diseases, e.g., hypervitaminosis D.