Eight genital isolates of Chlamydia trachomatis that each contained two different serovars were identified by typing with serovar-specific monoclonal antibodies. Mixed infections were confirmed by using serovar-specific monoclonal antibodies in an indirect immunofluorescence assay. For some isolates, fluorescein/rhodamine double-label indirect immunofluorescence with mixtures of IgM and IgG monoclonal antibodies to Chlamydia provided direct visual confirmation of mixed infection. Two isolates contained serovars D and F, and two E and F; one isolate contained serovars Ba and E, one D and J, one I' and F, and one E and J. In a sample of 352 genital isolates of C. trachomatis consecutively typed by dot ELISA with monoclonal antibodies, seven (2%) demonstrated mixed serovars.