An attempt was made to clarify the molecular characterization of zinc-induced bone protein synthesis in tissue culture. Calvaria were removed from weanling rat (3-week-old male) and cultured for periods up to 48 hr in Dulbecco's Modified Eagle Medium (high Glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. When calvaria cultured in the presence of 10(-5) to 10(-4) M zinc were pulsed with [3H] leucine, zinc caused a significant increase in the incorporation of [3H] leucine into the acid-insoluble residues of bone tissue. The soluble fraction obtained from cultured bone was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The major components in the fraction obtained from control bone were 68 killo-dalton (kDa) and 45 kDa proteins. These components were clearly increased by the presence of zinc (10(-4) M). The effect of zinc was completely abolished by the coexistence of 10(-6) M cycloheximide. Meanwhile, 10(-9) M estrogen or 10(-8) M insulin, which can stimulate bone formation, did not enhance the effect of zinc to increase bone 68 and 45 kDa proteins. The present findings suggest that zinc increases many bone protein components, especially 68 and 45 kDa proteins.