OBJECTIVE To evaluate the use of the Amplicor PCR assay in two European centres (Amsterdam and Vienna) as a standard for amplification assays in comparative studies. METHODS Both centres used the Amplicor PCR assay and their own standard diagnostic methods as the reference assay. Discrepant results were further analysed by an Omp1-PCR assay. In total 805 female patients and 614 male patients were included in the studies. RESULTS The prevalence of Chlamydia trachomatis infections in female patients was 10.0% in Amsterdam and 2.5% in Vienna and in male patients 13.7% and 10.8%, respectively. The mean sensitivity of the Amplicor PCR assay with cervical specimens from female patients was 92.5% and the mean specificity was 99.2%. For the reference assays the mean values were 87.5% and 100.0% respectively. The mean sensitivity of the Amplicor PCR assay with urine specimens from male patients was 70.3% and the mean specificity was 97.6%. For the reference assays these values were 64.9% and 100.0% respectively. The mean sensitivity of the Amplicor PCR assay with urethral specimens from male patients was 67.6% and the mean specificity was 98.5%. For the reference assays these values were 62.2% and 100.0% respectively. Of all specimens together, 1.0% showed an OD value between 0.25 and 0.5 in the Amplicor PCR assay and had to be retested. The sensitivity of the Amplicor PCR assay was less than could be expected from previous studies comparing amplification assays under similar conditions. Also, the sensitivity of the Amplicor PCR assay varied considerably between study centres using urine specimens from male patients-45.9% and 94.6%, respectively. CONCLUSIONS The Amplicor PCR assay cannot be considered as a standard for amplification assays in comparative studies.