Transforming growth factor-beta modulates the proliferation, differentiation, and synthetic activity of osteoblasts, but its mechanisms of action are not fully understood. Because ascorbate also influences osteoblast differentiation and is a cofactor for collagen synthesis, the present study examined the effect of transforming growth factor-beta on the initial rate of transport and steady-state concentration of ascorbate in an osteoblastic cell line. UMR-106 rat osteosarcoma cells accumulated reduced vitamin C from culture medium. Virtually all accumulation of ascorbate was accomplished by a saturable Na(+)-dependent transport mechanism. Transforming growth factor-beta increased the initial rate of ascorbate transport, measured in either attached or suspended cells. Within 24 h, the growth factor also increased the steady-state intracellular concentration of ascorbate, without significantly changing cell volume or the DNA or protein content of cultures. These data provide evidence that Na(+)-ascorbate cotransport activity controls ascorbate concentration in osteoblasts. Furthermore, the results indicate that both the transport rate and steady-state concentration of ascorbate in these cells are regulated by transforming growth factor-beta.