We recently reported the development of a chinchilla model of experimental otitis media (OM) that uses a pediatric clinical isolate of adenovirus type 1 (4) and in which an active infection with the wild-type strain was demonstrated. To expand upon these findings, this study was designed to determine whether we could demonstrate adenovirus infection-induced predisposition to bacterial OM in the chinchilla, as has been shown in human epidemiological studies (D. A. Clements, F. W. Henderson, and E. C. Neebe, p. 27-29, in D. J. Lim, C. D. Bluestone, J. O. Klein, D. J. Nelson, and P. L. Ogra, ed., Proceedings of the Fifth International Symposium on Recent Advances in Otitis Media, 1993; F. W. Henderson, A. M. Collier, M. A. Sanyai, et al., N. Engl. J. Med. 306:1377-1383, 1982). In addition, we were interested in determining whether altering the order of pathogen acquisition would further affect the outcome of disease incidence and severity. Toward this end, cohorts of chinchillas were inoculated intranasally with a strain of nontypeable Haemophilus influenzae (NTHi) (86-028NP) which colonizes the chinchilla nasopharynx but does not consistently induce culture-positive OM when inoculated intranasally (L. O. Bakaletz, T. M. Hoepf, D. J. Lim, and B. Tallan, Abstr. 90th Annu. Meet. Am. Soc. Microbiol. 1990, abstr. B-66, p. 37, 1990), adenovirus type 1 and then inoculated 7 days later with NTHi, NTHi and then inoculated 7 days later with adenovirus type 1, or both pathogens concurrently. All cohorts were observed over a 35-day period and assessed for incidence and severity of OM by several methodologies. The data collectively indicated that all animals receiving both pathogens developed OM of greater severity than those receiving only a single agent. Adenovirus inoculation followed 7 days later by NTHi inoculation was the order of pathogen acquisition which induced the most prolonged presence of NTHi in both the nasopharynx and the middle ear, the most severe tympanic membrane inflammation overall, and the most significant damage to and altered function of both middle ear and eustachian tube mucosae.