Nineteen independent hybrid cell lines that produce monoclonal antibodies to Chlamydia trachomatis surface antigens were prepared by the fusion of mouse myeloma cells with lymphocytes of mice that were immunized with C. trachomatis immunotypes B, C, and L2. Seven serologically distinct reaction patterns were detected by microimmunofluorescence (micro-IF) of elementary body (EB) preparations when culture fluids were tested against a panel of 18 chlamydial serotyping reference strains. These reaction patterns demonstrated genus-, species-, subspecies-, and type-specific distributions. Additionally, these antibodies were tested in parallel against reticulate body (RB) preparations of several chlamydial strains. Monoclonal antibodies that reacted with genus-specific antigens reacted preferentially with RB, whereas antibodies that reacted to species-, subspecies-, or type-specific antigens reacted equivalently to both RB and EB. Physiochemical characterization of antigens recognized by the different monoclonal antibodies was assessed by heat treatment, pronase digestion, periodate oxidation, and immuno-blot techniques. The genus-specific antigen was a heat-stable, pronase-resistant, and relatively periodate-sensitive component of less than 10,000 m.w. The species-, subspecies-, and type-specific antigens were heat stable, pronase sensitive, and periodate resistant. The antibodies that detected species- and subspecies-specific antigens predominantly reacted in immuno-blots with the 40,000 m.w. major outer membrane protein. These monoclonal antibodies now provide a new approach for the precise serologic classification and detection of different C. trachomatis strains.