Immunoneutralization studies with a monoclonal antibody to somatomedin C (Sm-C) were undertaken to further determine the role of this peptide in cellular proliferation. For our model we used density-arrested cultures of BALB/c 3T3 cells. Transient exposure of these cells to platelet-derived growth factor enables them to respond to platelet-poor plasma by progressing through the G1 stage and undergoing renewed DNA synthesis. In this system, the combination of nanogram concentrations of Sm-C and epidermal growth factor can fully substitute for plasma, and microgram concentrations of insulin can substitute for Sm-C by crossreacting with the Sm-C receptor. We now show that a monoclonal antibody to Sm-C, which in defined medium blocks the mitogenic effect of Sm-C but not insulin, also blocks the stimulation of DNA synthesis by human plasma or calf serum. Furthermore, by adding the antibody at progressively later times during G1, we show that these cells escape from their dependence on Sm-C for DNA synthesis after traversing G1 to a point at or near the G1/S boundary.