Chlamydia trachomatis infections are the most prevalent sexually transmitted diseases (STDs) in the United States. In acute-care settings such as clinics and emergency rooms, a desirable chlamydia screening assay should exhibit good sensitivity and good specificity and should provide test results while the patient is still present. The Biostar Chlamydia OIA (Biostar, Inc., Boulder, Colo.) is an optical immunoassay (OIA) that provides test results in less than 30 min and that uses a test format that allows office-based testing. This assay is performed entirely at room temperature without the need for rotators or other specialized equipment. The goal of this study was to compare the performance of the Biostar Chlamydia OIA for the detection of C. trachomatis with the performance of cell culture, direct fluorescent- antibody (DFA) assay (Syva MicroTrak; Syva Co., Palo Alto, Calif.), and PCR (Roche Amplicor Chlamydia trachomatis; Roche, Branchburg, N.J.) for the detection of C. trachomatis infections in women attending an urban STD clinic. For calculations of relative test performance (sensitivity, specificity, and positive and negative predictive values), patient specimens that yielded positive results by two or more of the four assays (cell culture, DFA assay, PCR, and OIA) were classified as 'true infections.' By these criteria, 42 of 306 total specimens were classified as positive for C. trachomatis (positive prevalence, 13.7%), 11 (3.6%; 10 by PCR and 1 by DFA assay) were positive by a single assay, and 253 (82.7%) were negative by all four tests. All culture-positive specimens were also positive by at least one other assay. Among the culture-negative specimens, 14 (5%) specimens were positive by two of the three non-culture-based assays used. By using the criterion that positivity by at least two of the tests indicated a true infection, the relative sensitivities were as follows: culture and PCR, 92.9% each; Biostar Chlamydia OIA, 73.8%; and DFA assay, 59.5%.