To determine limitations in commonly used methods for detection of Chlamydia trachomatis, 601 genitourinary specimens from patients in a sexually transmitted disease clinic were examined with quantitative cultures and by 2 different direct antigen tests, immunofluorescence (Micro Trak; Syva Company, Palo Alto, CA) and enzyme immunoassay (Chlamydiazyme; Abbott Laboratories, North Chicago, IL). Genital specimens were held no longer than 5 hours (at 4-degrees-C) prior to inoculation for culture; 28% (168/601) were positive. To evaluate the effect of storage on culture efficacy, duplicate specimens were also stored at -70-degrees-C and brought out subsequently for culture a second time. Only 32% (8/25) of specimens cultured within 5 hours and having <10 inclusions were positive on reculture, compared with 98% (49/50) positive for specimens with greater-than-or-equal-to 10 inclusions initially (P < 0.001). Sensitivities of the two antigen tests were similar and taken together diminished significantly (P < 0.001) as the number of organisms (inclusion forming units) in corresponding cultures decreased: 82% (51/62) sensitivity in cultures with >100 inclusions; 50% (22/44) with 10100 inclusions; and only 11% (6/53) with <10. Lack of urethral discharge in men with C. trachomatis infection (free of Neisseria gonorrhoeae) was associated with low numbers of inclusions (< 10) and antigen tests failed in 68% (15/22) of these patients.