Although serological methods or culture methods of Mycoplasma pneumoniae (Mp) from throat washings are used for diagnosis of Mp infection, the patient is often discharged from the hospital before definitive results can be obtained. Therefore we have developed a rapid detection from patients' throat smears by immunofluorescence with anti-rabbit IgG. Antibody specificity to Mp and the difference of titers between each lot have been a problem, because of polyclonal antibody which is prepared by immunizing a rabbit. Two hybridoma cell lines producing monoclonal antibodies to Mp were obtained by fusion of spleen cells from BALB/c mice immunized by Mp antigen with myeloma cells (X63-Ag8-6. 5. 3.). The high specificity to Mp of these monoclonal antibodies produced by the hybridomas was affirmed ELISA, immunofluorescence, and Western blot. A few cross-reactions were recognized by ELISA between Mp antigen and other Mycoplasma antigens. No cross-reactivity was found by indirect immunofluorescence. Throat smears from 4 patients suffered from Mp infection were examined by immunofluorescence with monoclonal antibody. Specific immunofluorescence was seen in all smears. Both granular and diffuse types of fluorescence were seen in smears from case 1 and 2. Only diffuse type was found in smears from case 3 and 4. Monoclonal antibodies can be obtained at any time and amount. The method may be useful and convenient for the rapid diagnosis of Mycoplasmal pneumonia.