We examined the effects of insulin on bone formation including the mineralizing process. Twenty-day fetal rat parietal bones were cultured for 96 h on grids in a serum-free medium. For the precise assessment of bone formation, histomorphometry, with an image analyzing system, was used to measure the areas of mineralized bone and bone matrix, and the numbers of osteoblasts and osteoclasts. In order to confirm the effects of insulin on bone mineralization, the calcium content of the bone and the release of previously incorporated 45Ca into the medium were measured. Insulin, at a concentration of 10-6 M or higher, increased the areas of mineralized bone and bone matrix, and the number of osteoblasts. Osteoclasts were seldom observed in bones on day 0 or in bones treated with insulin. In bones treated with insulin at a concentration of 10-6 M, the calcium content of bone increased. At an insulin concentration of 10-'M or higher, the dry weight of decalcified bone increased. Lactate production in the medium increased dose-dependently. The inhibited release of 45Ca in bones indicated that insulin acts by increasing calcium retention. We demonstrated that insulin has an effect on bone-forming and bone-resorbing cells to enhance the bone forming process from matrix formation to mineralization.