Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.