To evaluate a commercially available DNA hybridization test, the Gen-Probe Pace 2 assay (GP) was compared with the Chlamydiazyme (CZ) for the detection of Chlamydia trachomatis in urethral and endocervical specimens. Samples were collected from 300 persons, including 199 registered and 43 nonregistered female prostitutes, examined for screening purposes, as well as 22 male and 36 female symptomatic sexually transmitted disease (STD) patients. The overall prevalence of C. trachomatis was 7.3% in all persons examined with an infection rate of 20.9% in the nonregistered prostitutes, 2.5% in the registered prostitutes and 13.8% in the STD patients. The overall concordance of both diagnostic methods was 98.7% in all samples examined, 100% in male and 98.6% in female samples. Specimens with discordant results were further analyzed by a direct immunofluorescence test (MicroTrak) and by the probe competition assay (PCA). All samples only positive in the GP assay could be confirmed by the PCA while one result which was positive in the CZ could not be confirmed by any other test. The GP assay was superior to the CZ, when compared with true-positive and -negative results. The data demonstrate that the GP assay can be recommended as an alternative diagnostic technique to the CZ for Chlamydia diagnosis.