Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.