Somatomedin-C/insulin-like growth factor-I (Sm-C/IGF-I) and insulin stimulate DNA synthesis and cell replication in cultured human fibroblasts. It has been postulated that the growth-promoting actions of both peptides are mediated through the type I Sm-C/IGF-I receptor. This study tests this hypothesis using two recently developed monoclonal antibodies. The antibody designated sm 1.2 is directed to Sm-C, whereas the antibody designated αIR-3 is directed against the type I receptor for Sm-C/IGF-I. Radiolabeled monoclonal antibody αIR-3 was bound to human foreskin fibroblasts in a reversible time-dependent fashion, with 90% of the specific binding complete after 6 h of incubation at 15 C. Binding of [125I]aIR-3 was ompletely inhibited by excess unlabeled antibody, but not by 50 nM Sm-C or 1000 nM insulin. Specific binding of [125I]Sm-C fell to 27% of the control value in the presence of 50 nM aIR-3, and this concentration of antibody significantly reduced the mitogenic response to both Sm-C and insulin. Antibody sm 1.2 blocked the mitogenic response to exogenous Sm-C, but did not block the response to insulin; indeed, in some experiments, sm 1.2 enhanced the response to insulin. We postulate that this enhancement is the result of neutralizing endogenously produced Sm-like substances. This study provides further evidence that the growth-promoting effects of insulin in this cell type are the result of interaction with the Sm-C/IGF-I receptor.