From April to September 1993, 305 men and 447 women in Hamilton, Canada, consented to the collection of a urethral or cervical swab, respectively, for culture and 20 ml of first-void urine (FVU) for testing by the enzyme immunoassay Chlamydiazyme and by ligase chain reaction (LCR) in the form of a kit from Abbott Laboratories called LCx Chlamydia trachomatis. Evaluation of test performance with each specimen was calculated on the basis of an expanded "gold standard" of a patient found to be positive by culture or by a confirmed nonculture test. By using this expanded standard, the prevalence of infection was determined to be 6% (27/447) for the women and 18.4% (56/305) for the men. LCR testing of FVU in both studies was the most sensitive approach (96%). The performance of Chlamydiazyme was as follows: cervical swab, 78.3% sensitivity; female FVU, 37% sensitivity; and male FVU, 67.9% sensitivity. Culture was the least sensitive approach to diagnosis: female cervix, 55.6%; and male urethra, 37.5%. LCR testing of FVU from men or women diagnosed the greatest number of genitourinary tract infections with no false positives.