The potentiation of RSV lower respiratory tract disease by prior inoculation of formalin-inactivated RSV vaccine which occurred during clinical trials of the vaccine 20 years ago was reproduced in the laboratory for the first time. Potentiation of pulmonary pathology was observed when cotton rats (Sigmodon hispidus) previously immunized with formalin-inactivated RSV vaccine underwent a RSV infection of their respiratory tract. Within 24 hours after infection with RSV, immunized cotton rats developed exaggerated pulmonary lesions that reached a maximum by day 4. Histologically, the lesions resembled an experimental pulmonary Arthus reaction. An action of formalin on RSV appears to be responsible for this effect, because live virus or virus heated in the absence of formalin did not induce enhanced immunopathology. Epitopes on the fusion (F) and/or attachment (G) RSV surface glycoproteins involved in inducing neutralizing antibodies were modified by formalin resulting in a reduction or ablation of a neutralizing antibody response. Nonetheless cotton rats inoculated parenterally with formalin-inactivated virus developed a high level of F and G antibodies measurable by an enzyme-linked immunosorbent assay indicating that most of the epitopes not involved in neutralization were not altered by formalin. At this time, the effect of formalin on RSV cannot be localized to either the F or G glycoprotein of RSV. Staining of lung sections from vaccinated, infected animals showed abundant deposits of IgG and C3, indicating a major role for a type III (Arthus) immunopathologic reaction. Subsequent studies in two other species of cotton rats (S. fulviventer and S. arizonae), one inbred strain of mice (DBA/2N) and owl monkeys have all shown a similar pattern of disease enhancement in animals pretreated with formalin-killed virus.