The APTIMA COMBO 2 assay, which detects and amplifies rRNA from Chlamydia trachomatis and/or Neisseria gonorrhoeae, is approved for use on ThinPrep liquid-based Pap test specimens. The objective was to determine the clinical utility of the APTIMA assays (APTIMA COMBO 2 assay, APTIMA CT assay for Chlamydia trachomatis, and APTIMA GC assay for Neisseria gonorrhoeae) for screening women during their annual Pap exam, using SurePath liquid-based Pap test specimens. Two cervical samples were collected from 1,615 females attending six clinical sites in North America. A cervical broom sample was processed for cytology, with the residuum aliquoted into an APTIMA specimen transfer kit tube. The second cervical swab sample was put into APTIMA specimen transport medium, and both samples were tested with each APTIMA assay on a direct sampling system. Using a subject-infected status that utilized cervical-swab specimen results from two APTIMA assays, the prevalence was 7.9% for Chlamydia trachomatis and 2.5% for N. gonorrhoeae. For the liquid-based Pap samples, the sensitivities, specificities, positive predictive values, and negative predictive values for Chlamydia trachomatis detection were 85.2%, 99.5%, 93.2%, and 98.7%, respectively, for the APTIMA COMBO 2 assay and 89.1%, 98.7%, 85.7%, and 99.1%, respectively, for the APTIMA CT assay. For N. gonorrhoeae detection, the values were 92.5%, 100%, 100%, and 99.8%, respectively, for the APTIMA COMBO 2 assay and 92.5%, 99.9%, 97.4%, and 99.8%, respectively, for the APTIMA GC assay. The high predictive values support the use of the assays with SurePath liquid-based Pap specimens processed with the APTIMA specimen transfer kit. Copyright © 2007, American Society for Microbiology. All Rights Reserved.