Otitis media was produced in chinchillas by right-sided intrabullar inoculation with nontypable Haemophilus influenzae, and susceptibility to reinfection was investigated. After resolution of initial right-sided infection, animals underwent ipsilateral or contralateral intrabullar rechallenge with the same strain. After ipsilateral rechallenge right ears were completely protected against reinfection; previously uninfected left ears were similarly protected on contralateral rechallenge. Previously infected ears remained fully susceptible to infection with a heterologous strain of nontypable H. influenzae. Using an enzyme-linked immunosorbent assay, we measured the serological response to outer membrane protein and lipopolysaccharide antigens during initial infection. A greater than or equal to 10-fold rise in titer of antibody to homologous outer membrane proteins was observed in all 11 animals tested. Most animals exhibited a minimal serological response to lipopolysaccharide. Thus experimental otitis media due to nontypable H. influenzae induces strain-specific protective immunity and a concomitant serological response to outer membrane proteins.