The aim of this study was to evaluate the performance of an improved sample preparation procedure that enhances the sensitivity of the Chlamydia trachomatis Cobas Amplicor assay (Roche, Switzerland). This procedure was developed after it was observed that, in some cases, endocervical swabs positive for Chlamydia trachomatis in a direct immunofluorescence assay were negative in the Chlamydia trachomatis Cobas Amplicor. For this procedure, the initial sample volume was increased over that recommended by the manufacturer, the sample was concentrated by centrifugation, and the yield of the extraction was increased by an additional enzymatic lysis. Five hundred sixteen endocervical swabs from women visiting a family planning clinic were tested in parallel by the Chlamydia trachomatis Cobas Amplicor using the extraction procedure recommended by the manufacturer and the modified in-house sample preparation procedure. Eight samples were positive with both procedures. Seven additional samples were positive with the in-house method only. The results show that the in-house sample preparation procedure markedly improves the test sensitivity of the Chlamydia trachomatis Cobas Amplicor assay. Consequently, the use of this improved screening protocol will facilitate the detection of even low-level infections with Chlamydia trachomatis, which in turn will lead to the introduction of earlier therapeutic interventions.