Purpose: To study whether acute otitis media caused by encapsulated or nontypeable Haemophilus influenzae confers cross-reactive protective immunity in an animal model system and to explore the possible involvement of various humoral specific antibodies in protection.Materials and Methods: Rats were intrabullarly challenged with H influenzae type b and two different nontypeable H influenzae strains. One month after the initial infection, the animals were rechallenged ipsilaterally or contralaterally with either a homologous or heterologous strain, and the susceptibility to reinfection was investigated by otomicroscopy.Results: The animals challenged and rechallenged with the type b strain were well-protected ipsilaterally and contralaterally, while the protection after homologous rechallenge with a nontypeable strain was partial in the ipsilateral ear and very poor in the contralateral ear. Middle ears previously infected with a nontypeable strain remained fully susceptible to infections with heterologous strains, but there was an indication of cross-protection in the animal groups where the first episode of acute otitis media was caused by type b and the second by a nontypeable strain. Using the Western blot technique and an enzyme linked immunosorbant assay, the serological response to different outer membrane proteins, especially protein D, of H influenzae during and after middle ear infection were investigated. The serological response from the type b infected animals were generally more distinct, while the antibody levels against protein D were lower in these groups compared with the groups infected with nontypeable strains.Conclusions: These data indicate that H influenzae type-b-induced experimental otitis media results in a better protection than a nontypeable-induced, and H influenzae b confers a cross protection.