In secondary middle ear immune response, kinetics of immunocytes, especially T cell subsets, was examined in the round window membrane (RWM) using immunohistochemical methods. Healthy BALB/c mice and keyhole limpet hemocyanin (KLH) antigen were employed in this study. The inflammatory responses of the RWM and middle ear were investigated after antigen challenge into the middle ear bulla. We used antibodies against murine macrophages and granulocytes (anti-Mac-1), murine helper/inducer T cells (anti-Lyt-1 and -L3T4), murine suppressor/cytotoxic T cells (anti-Lyt-2), murine interleukin-2 receptor (7D4), murine immunoglobulins (anti-IgG, -IgM and -IgA) and KLH. In the RWM and middle ear mucosa, inflammatory cells were observed at 6 hours, peaking on days 3-7, whereas these cells were rarely seen in the scala tympani of the basal turn. Luminal effusion with an enormous infiltration of inflammatory cells, which consisted mainly of Mac-1 cells, IgG cells and IgM cells, was observed in the middle ear cavity on days 1-7 post antigen challenge. In the inflamed RWM, Mac-1 cells were the predominant cell type followed by helper T cells, interleukin 2 receptor positive cells and IgG positive cells, though IgM, IgA and Lyt-2 positive cells were rarely observed after antigen challenge. Our results suggest that RWM has the ability to protect inner ear by cellular immune response through activated helper T cells and Mac-1 cells.