The middle ear mucosal system and the humoral systemic immune factors are the two immunological systems whose involvement in the defence against acute otitis media (AOM) have been most intensively studied. However, their respective importance and their mutual influence is not clear. In the present study, a rat model for pneumococcal AOM was used to further elucidate the involvement of systemic immunity in protection against pneumococcal AOM. Six groups of male Sprague-Dawley rats were immunised with pneumococcal vaccine (PneumovaxRN) or live pneumococci (type 3) via one of three different routes: intraperitoneally, into the gastrointestinal tract (GIT) or into the right middle ear. A subsequent middle ear challenge (re-challenge in one group) with the same pneumococcal strain was performed after 4 days to 8 weeks in the different groups. Systemic immunity was found to be triggered, not only by systemic immunisation, but also by antigenic stimulation of the mucosa in the middle ear and in the GIT. In all groups but that immunised in the GIT, no new peak of specific IgG antibody response was demonstrated in serum after middle ear challenge/re-challenge. In contrast, half of the rats immunised in the GIT showed such a response not only after the inoculation into the GIT but also after a later performed middle ear challenge. Though a faster resolution of pus from the middle ear was observed in rats from all but one group, a significant reduction in the number of rats who developed AOM occurred exclusively among those rats that had previously manifested serological response to immunisation in the GIT.