Newcastle disease virus (NDV)-infected HeLa and FL cells showed small polykaryocytes at about 24 h postinfection, while the addition of anti-FL-cell rabbit, rat, or mouse serum to the NDV-infected cells gave rise to giant polykaryocytes at 15 h postinfection. We isolated three monoclonal antibodies (MAbs) (4-5-1, 6-1-13, and 7-2-1) capable of enhancing giant polykaryocyte formation in NDV-infected HeLa cells. These MAbs immunoprecipitated gp80 or gp135, which were detected mainly on the surface of HeLa cells. A functionally intact F protein was essential for antibody-enhanced cell fusion, and hemagglutinating (receptor-binding) activity of HN protein was involved in the fusion at an early stage; that is, the MAbs enhanced NDV-mediated syncytium formation. These molecules were considered to have the ability to regulate NDV-mediated cell fusion and thus were designated fusion regulation protein (FRP)-1 (gp80) and FRP-2 (gp135). Anti-FRP MAbs enhanced the susceptibility of cells to fusion activity of NDV. Anti-FRP-1 MAbs reacted with a molecule on the surface of every cell derived from humans and monkeys but showed no cross-reactivity with mouse or hamster cells. FRP-2 could be detected in limited cell lines of human origin.