A quantitative analysis of immunocompetent cells in the middle ear mucosa of mice was carried out by an indirect immunostaining method using various monoclonal antibodies. Mice bred in germ-free, specific pathogen-free, and conventional conditions were used to examine nonimmunized middle ear mucosa. Middle ear mucosae of otitis media-induced mice were also examined. In normal middle ear mucosa, mast cells were substantial, followed by Mac-1-positive cells and lymphocytes. Even though IgA-, IgM-, and Lyt-1-positive cells were seen in the mucosa of conventional mice, IgM-positive cells were seen only in mucosae of specific pathogen-free and germ-free mice. In otitis media-induced mice by inoculation with nontypable Haemophilus influenzae or lipopolysaccharide, Mac-1-positive cells were dominant. Although the numbers of IgM- and Lyt-1-positive cells increased markedly, the numbers of other lymphocyte subsets did not increase until 14 days after inoculation. These findings suggest that the middle ear is immunologically a potential organ as long as it is not exposed to antigenic stimulation. It is considered to be an immunoreactive site only after it has been activated with pathogens.