Chlamydia trachomatis and Neisseria gonorrhoeae are common causes of sexually transmitted infections, and there is interest in screening SurePath liquid-based Pap (L-Pap) samples with Aptima Combo 2 (AC2), Amplicor (AMP), and ProbeTec ET (PT) assays. SurePath L-Pap samples and a cervical swab (CS) were collected from 394 women attending health clinics in Hamilton and Toronto, ON, Canada. L-Pap samples were tested with the three assays prior to being processed for cytology, and the CS sample was tested with AC2. The prevalence of C. trachomatis was 8.9%, and that of N. gonorrhoeae was 1.5%. By using the positives from CS testing, as well as CS negatives corresponding to L-Pap samples that tested positive in 2 of 3 assays, the sensitivities of AC2, AMP, and PT for C. trachomatis in precytology samples were calculated to be 97.1% (34 of 35 positive samples were detected), 91.4% (32 of 35 were detected), and 77.1% (27 of 35 were detected), respectively. Six women were infected with N. gonorrhoeae. After cytology processing, the results of testing the remaining liquid in the L-Pap vial and the cell-enriched fraction for C. trachomatis by AC2 showed positive agreements of 98.9% (kappa [k], 0.93) and 98.7% (k, 0.92), respectively, with the results of testing precytology L-Pap samples. Although all testing showed high specificity, testing for C. trachomatis by AC2 was significantly more sensitive than testing by PT for SurePath samples (P = 0.02). Newer versions of AMP (Cobas 4800) and PT (Q(x) with XTR technology) need published evaluations for detecting C. trachomatis and N. gonorrhoeae in L-Pap samples. C. trachomatis testing can be performed with similar results on pre- and postcytology SurePath samples.