The technical complexity of determining the serovar of Chlamydia trachomatis strains has limited the use of serotyping in clinical and epidemiologic studies. We developed a simple method for rapidly serotyping isolates of C. trachomatis by using monoclonal antibodies in a dot-enzyme-linked immunosorbent assay (ELISA) system. Isolates were passaged three to six times in shell vial cultures to greater than 50% monolayer infection, and chlamydial elementary bodies were isolated by sonication and microcentrifugation. Chlamydial antigen was spotted onto a series of replicate nitrocellulose membrane patches and reacted with C. trachomatis-specific monoclonal antibodies. Bound antibody was detected visually by a color reaction by using peroxidase-conjugated anti-mouse immunoglobulins. This method can be routinely applied to 60 or more specimens concurrently. We compared dot-ELISA serotyping with monoclonal antibody microimmunofluorescence serotyping of 124 clinical C. trachomatis isolates and found that dot-ELISA has sensitivity and serotyping accuracy comparable to that of monoclonal antibody microimmunofluorescence.