It has been shown previously that MCF-7 cells proliferate in response to nanomolar concentrations of IGF-I and IGF-II. It has also been reported that the actions of both peptides are mediated through the IGF-I receptor. To further characterize these observations, we used MCF-7 and Hs578T cell lines in the serum-free/phenol red-free system developed by Ogasawara and Sibarsku, 1988. Cell proliferation was studied in the presence of insulin, IGF-I and -II and a series of growth factor receptor antibodies. No effect was observed on Hs578T cell proliferation with any of the growth factors. However, MCF-7 cells were stimulated 4-5 fold with IGF-I and insulin, while IGF-II was only slightly less potent. alpha IR3, a monoclonal antibody directed against the IGF-I receptor, was stimulatory when added alone. However, alpha IR3 blocked approximately 50% of the IGF-I response, only 5% of the insulin response, and did not block the IGF-II effect on cell proliferation. These data suggest that alpha IR3 and IGF-I are acting as agonists through the IGF-I receptor, but that insulin and IGF-II are acting through other receptors. Two different IGF-II/M-6-P receptor antibodies and an insulin receptor antibody failed to significantly block IGF-II actions. All three antibodies were stimulatory when added alone. beta-gal inhibited 27% of the IGF-II response and had no effect when added alone. Since beta-gal decreases the binding affinity of the IGF-II/M-6-P receptor for IGF-II and does not bind to the IGF-I or insulin receptor, these data suggest the possibility that IGF-II mitogenic action is mediated through the IGF-II/M-6-P receptor. In summary, these data indicate that nanomolar concentration of insulin, IGF-I and IGF-II are potent mitogens in MCF-7 cells and can potentially stimulate cell proliferation through all three receptors.