Studies were conducted with the goal of developing a kit for assaying anti-Chlamydia pneumoniae antibodies in human serum which would enable judging positive cases with high specificity by means of an objective numerical index. Thus, an enzyme-linked immunosorbent assay (ELISA) method employing a C. pneumoniae outer membrane complex protein was established. Elementary bodies (EB) were purified from the YK-41 strain of C. pneumoniae, and subsequent treatment with Sarkosyl, DNase and RNase yielded chlamydial outer membrane complex (COMC). COMC was employed as the antigen and immobilized on 96-well microplates for ELISA method. This ELISA method was used to test 51 serum specimens from patients who had been demonstrated to be positive for C. pneumoniae antigen (throat swab: PCR positive), and the levels of IgG, IgA and IgM antibodies were assayed. For each specimen, comparison was made with the antibody titers determined by the micro immunofluorescence test (Micro-IF method). The results showed good correlation coefficients of 0.950 for IgG, 0.852 for IgA and 0.866 for IgM. In addition, the two assay methods showed the following high agreement rates: 90.2% for IgG, 84.3% for IgA and 82.4% for IgM. Specimens which did not yield the same results with the ELISA method and Micro-IF method were subjected to analysis by Western blot method, and the rates of agreement with the ELISA results were 80% for IgG, 87.5% for IgA and 88.9% for IgM. These data indicate the efficacy of this new ELISA method. Moreover, COMC was reacted with mouse antisera to three Chlamydia species, and the mouse IgG antibody was assayed. Anti-C. pneumoniae antiserum showed the strongest reactivity, whereas weaker reactivity was shown by anti-C. trachomatis antiserum (1/32nd of the reactivity of the anti-C. pneumoniae antiserum) and anti-C. psittaci antiserum (1/4th). In addition, sera from patients infected with C. trachomatis or C. psittaci (Psittacosis) were subjected to the ELISA method using COMC from C. pneumoniae. It was found that the correlation between the ELISA and Micro-IF methods was higher in relation to the anti-C. pneumoniae antibody titer than either the anti-C. trachomatis antibody titer or anti-C. psittaci antibody titer. These findings indicate this new assay kit based on the ELISA method has high specificity for C. pneumoniae.