An established pulp cell line (RPC-C2A) was used to study the regulatory effect of insulin on dentinogenesis. Insulin increased alkaline phosphatase activity and the incorporation of [2,3-3H]-proline into collagenase-digestible protein, whereas [3H]-thymidine incorporation by the cells was inhibited by insulin. The enhancing effect of insulin on alkaline phosphatase activity was inhibited by epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta). The stimulatory effect of insulin on collagen synthesis was also inhibited when insulin was combined with EGF, but was accelerated by the addition of TGF-beta. Inhibitory effects of insulin on the [3H]-thymidine incorporation were potentiated by EGF, though EGF alone strongly increased the effect; whereas the addition of TGF-beta had no significant effect on the insulin action. These findings suggest that insulin may be concerned with the differentiation of pulp cells in dentinogenesis and that EGF or TGF-beta regulate the insulin effects.