A procedure for use of the Amplicor Chlamydia PCR with the Syva MicroTrak enzyme immunoassay (EIA) medium was developed, and the performance of the Syva MicroTrak EIA was evaluated by use of PCR and the Syva MicroTrak direct immunofluorescence assay (DFA) as confirmatory methods. PCR detected Chlamydia organisms at a 10-fold greater dilution than did DFA. Of 366 specimens, 119 specimens were positive by both PCR and DFA, 6 specimens were positive only by PCR, and 241 specimens were negative by both PCR and DFA. Subsequently, DFA and the developed PCR procedure were used prospectively for confirmation of EIA results in a defined negative gray zone between the cutoff value and 30% of the cutoff value (70% below the cutoff value). All specimens with results above the EIA cutoff value were also subjected to confirmation with DFA and PCR. EIA was performed on 7,748 endocervical swab specimens, of which 494 (6.4%) were subjected to confirmation, and on 968 male urethral swab specimens, of which 185 (19.1%) were subjected to confirmation. A "gold standard" was based on the findings by DFA and PCR, and divergent results were resolved by a major outer membrane protein-based PCR. Forty-five of 160 female specimens (28.1%) and 11 of 93 male specimens (11.8%) within the defined negative gray zone were found to be positive. Of 334 female specimens having absorbance unit (AU) values above the EIA cutoff value, 258 could be confirmed, thereby giving a positive predictive value of 77% (258/334). Accordingly, the positive predictive value with male specimens was 95% (87/92). The prevalence of Chlamydia trachomatis-positive specimens was 3.9% (303/7,748) in females and 10.1% (98/968) in males. All male specimens having AU values above 1.0 in the EIA were confirmed positive. In contrast to this, 16 females with AU values above 1.0 in the EIA could not be confirmed positive with either DFA or PCR. The mean age of these females was higher than that of patients testing negative for C. trachomatis (P < 0.005). This might suggest an age-dependent change in vaginal colonization with an organism(s) crossreacting in the EIA. Thus, the PCR procedure developed can be used for confirmation of EIA results, testing specimens with AU values in the defined negative gray zone improves the sensitivity of EIA, and all specimens testing positive in EIA should be subjected to confirmation.