Ocular swabs from 30 consecutive patients with follicular conjunctivitis were screened for Chlamydia trachomatis, herpes simplex virus, and adenovirus by a variety of laboratory techniques. For the detection of C. trachomatis, we compared two polymerase chain reaction methods, McCoy cell culture isolation, and the direct fluorescein-conjugated monoclonal antibody test. Four of 30 patients tested positive for C. trachomatis by using both conventional and biotinylated polymerase chain reaction methods. Two of the four patients were also McCoy cell culture-positive for C. trachomatis and one of four patients tested positive by using a fluorescein-conjugated chlamydial monoclonal antibody test. All four patients responded to oral antibiotic treatment. On follow-up testing, all four patients were polymerase chain reaction-negative, McCoy cell culture-negative, and fluorescein-conjugated antibody test-negative for C. trachomatis. The polymerase chain reaction appears to be an equally specific and more sensitive method than McCoy cell culture or fluorescein-conjugated antibody testing for the detection of C. trachomatis from ocular specimens.