A fibroblast growth regulator, which inhibits the growth and division of proliferating fibroblasts, has been isolated from medium conditioned by exposure to density-inhibited mouse 3T3 cells. This partially purified preparation of the growth-inhibitory activity, termed FGR-s, contained two major polypeptides (Mrs, 10,000 and 13,000). Using FGR-s as the immunogen, we have carried out in vitro immunization of rat splenocytes and have generated hybridoma lines, each secreting an antibody directed against components of the FGR-s preparation. One such monoclonal antibody, designated 2A4, specifically bound the Mr 13,000 polypeptide of FGR-s. Antibody 2A4 also neutralized the growth-inhibitory effect of FGR-s in a concentration-dependent fashion. These results strongly suggest that the Mr 13,000 polypeptide carries growth-inhibitory activity.