Palivizumab (PZ) is the only monoclonal antibody currently available for use in humans against an infectious disease. PZ is administered prophylactically for respiratory syncytial virus (RSV) infections. RSV selected in cell culture for growth in the presence of PZ develops F gene mutations and can be resistant to PZ prophylaxis in cotton rats. Here, we evaluated the potential for PZ-resistant RSV mutants to arise in vivo. Cotton rats were immunosuppressed with cyclophosphamide, administered PZ, and inoculated intranasally with RSV. Lungs were harvested 12 weeks after RSV infection, reverse transcription-PCR-amplified F gene fragments were cloned into plasmids, and the nucleotide sequences of the cloned cDNAs were determined. Three of the five animals had mixed populations of lung virus, and over 50% of the clones from the three animals revealed F gene mutations associated with resistance to PZ. A virus completely resistant to PZ neutralization was recovered from the lung homogenate of a rat that had received PZ. Thus, prolonged pulmonary replication of RSV in the presence of PZ was followed by the appearance of viruses resistant to PZ. The potential for the development of resistance is a consideration as the antibody is used prophylactically against RSV and as passively administered antibodies are under development for other infections, including emerging viruses and agents of biodefense.Respiratory syncytial virus (RSV) is the principal cause of viral respiratory infections among infants and young children and causes disease in adults, with the elderly at particular risk for severe disease (8,14,26). In addition, immunocompromised patients may suffer serious morbidity and even mortality due to RSV infections (13, 16). Palivizumab (PZ), is the first commercially available antibody for use against an infectious disease. PZ, a humanized neutralizing monoclonal antibody reactive with an epitope on the F glycoprotein of RSV, is used prophylactically for high-risk children with preterm birth or underlying cardiorespiratory disorders (1).RSV escape mutants from monoclonal and polyclonal antibodies against the F and G proteins have been derived in cell culture (5,9,15,23,31,33). We selected RSV resistant to PZ by replication of virus in the presence of PZ in cell culture (36). Point mutations occurred at two sites in the F gene. At positions 828 (A-T, virus MP4) and 827 (A-C, virus MS412) changes resulted in two different amino acid changes at position 272 in the F1 subunit (Lys to Met or Gln, respectively). Both changes were associated with resistance to PZ neutralization in vitro. In addition, viruses with these point mutations were completely resistant to the prophylactic effects of PZ in cotton rats. A point mutation at another site, 816 (A-T), led to an amino acid substitution from Asn to Ile at position 268 in the F1 subunit. This virus, F212, was partially resistant to PZ neutralization but remained fully susceptible to PZ prophylaxis (15 mg/kg of body weight) in cotton rats. Interestingly, F212 grew to lower t...