Centrifugation of the cells in low concentrations (1%) of fetal bovine serum caused a 1.9-fold increase of [³H] thymidine incorporation on day 3 from the start of centrifugation, and gradually decreased with culture up to day 9. Alkaline phosphatase activity was not affected by centrifugal force until day 5, and increased rapidly after day 7. Stimulation of DNA synthesis by centrifugation was abolished in the presence of H-7, an inhibitor of protein kinase C. These results suggest that centrifugal force stimulates the proliferation of osteoblastic cells through an autocrine secretion of some diffusable growth-promoting activity. Additional centrifugation of the cells also slightly stimulated alkaline phosphatase activity, although this did not directly influence the cell's osteocalcin-production activity.