This study reports the establishment of cloned hybridomas between mouse myeloma cells (P3-X63-Ag8, 6.5.3) and spleen cells from BALB/c mice immunized with lung homogenates of nude mice (BALB/c nu/nu) heavily infected with Pneumocystis carinii. A hybridoma subclone, designated 1E12-8, produced antibodies of an IgG1 subclass. Using indirect immunofluorescent antibody techniques, the monoclonal antibody was found to be directed toward the pellicle antigen of air-dried P. carinii both in trophozoite and cyst forms, and to recognize the P. carinii antigen not only from nude mice but also from rats. The antibody did not cross-react with other components of the infected host lung tissue and showed little cross-reactivity with fungi examined. The monoclonal antibody should be useful in the isolation and identification of corresponding antigenic substances of P. carinii and may provide a useful tool in epidemiology, taxonomy, and diagnosis of this organism.