The insulin-like growth factors (IGF)-I and -II are bound to specific carrier proteins in the circulation. For investigation of their physiological role, the acid-stable subunit of the major binding protein (SmBP) was isolated from human plasma Cohn fraction IV. Its effect on the mitogenic activity of IGF-I was studied with baby hamster kidney fibroblasts (BHK-21) and human skin fibroblasts. While free IGF-I had no effect on thymidine incorporation into DNA with BHK-21 cells and only a moderate effect with human fibroblasts under standard conditions, DNA synthesis was significantly enhanced with both cell lines if IGF-I was complexed with SmBP before the experiment. The enhancement was optimal at an approximately equimolar ratio of both peptides. In contrast to experiments in which large concentrations of IGF-I were added at the beginning, repeated addition of small quantities of free IGF-I at hourly intervals clearly stimulated DNA synthesis in BHK-21 cells. Binding studies with radiolabeled SmBP revealed no evidence for direct interaction with either cell line. It is concluded that SmBP acts as a reservoir, releasing continuously low amounts of IGF-I and thereby creating a steady state situation of receptor occupancy, which appears to be a better mitogenic stimulus than temporary large concentrations of IGF-I.