Objectives: To assess the reliability of different laboratory methods for the detection of Chlamydia trachomatis in rectal specimens Methods: 1782 rectal specimens confirmed as C trachomatis positive using a standard laboratory method, were forwarded to the Sexually Transmitted Bacteria Reference Laboratory (STBRL). All specimens were retested using a C trachomatis specific independent in-house real time polymerase chain reaction (PCR). If this test was negative, a second test (Artus Real-Art PCR Kit) was employed as a confirmation. A correlation between real time PCR results obtained at the reference centre (STBRL), and the method of C trachomatis detection used in the primary laboratory was undertaken. Results: The percentage of specimens that could be confirmed as positive, compared with primary method of detection was as follows: C trachomatis culture 87.5%, strand displacement assay (SDA: Becton Dickinson) 93.4%, Cobas Amplicor (Roche) 89.2%, Aptima Combo Two assay (Genprobe) 83.3%, and enzyme immunoassays (EIA) 35.4%. Conclusions: High rates of confirmation can be achieved using an independent real time PCR assay to examine rectal specimens which had initially tested C trachomatis positive using nucleic acid amplification tests and chlamydia tissue culture. This is not possible for specimens that had been screened using EIA tests, which reflects the low specificity of this test when used for rectal specimens. Laboratories currently using EIA based assays to test rectal specimens should review this approach.