A formalin-inactivated respiratory syncytial virus vaccine was used to immunize infants in the mid-1960s; when these children later were naturally infected by the virus they developed markedly accentuated disease, and two died. For the present work, a new batch of vaccine was prepared using the original formula. Administration of either the old or new vaccines resulted in enhanced lesions in immunized cotton rats subsequently challenged with live virus, although administration of the vaccine reduced virus replication by 90%. Animals primed with formalin-inactivated virus and challenged developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which appear to be specific markers for the vaccine enhancement. The newly prepared vaccine appears suitable as a reference standard for studying the mechanism of vaccine-enhanced disease caused by this virus. Additionally, we reviewed the lesions in the lungs of the two humans who died with the vaccine-enhanced disease in 1967, and found that they were similar to, but more severe than those seen in the cotton rats.