Somatomedin-C (SM-C) is a growth hormone-dependent polypeptide with potent mitogenic activity in vivo and in vitro. In the present study, we show that four human breast cell lines maintained in long-term tissue culture (MCF-7, T47-D, MDA-MB-231, and HBL-100) have type I somatomedin receptors and that SM-C stimulates DNA synthesis in these cells. The concentration of SM-C required for half-maximal stimulation of DNA synthesis varied from 0.03 nM in the MCF-7 cell line to 0.6 nM in the T47-D cells. Porcine insulin also stimulated DNA synthesis in all cell lines but, compared to SM-C, 10- to 1000-fold-higher concentrations were required. SM-C receptors on the four breast cell lines were characterized by competitive binding and chemical cross-linking techniques. The four cell lines varied widely in their SM-C binding. In three of the cell lines (MCF-7, MDA-MB-231, and HBL-100), the SM-C receptor had a Kd for SM-C of 0.5 to 1 nM, and insulin competed for binding but with a potency 1/10 to 1/100 that of SM-C. In the T47-D cell line, the Kd for SM-C binding was 4 nM, and insulin competed poorly for binding. Chemical cross-linking studies showed that all four cell lines have type I somatomedin receptors. Variations in the sensitivity to SM-C and insulin stimulated DNA synthesis in the MCF-7 and T47-D cell lines correlated with type I somatomedin receptor binding by these cells. The data indicate that SM-C is mitogenic for cultured human breast cells and are consistent with the hypothesis that the mitogenic effect of insulin for these cells is mediated through the type I somatomedin receptor.