The effect of triaging women with atypical squamous cells of undetermined significance (ASC-US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high-grade disease. We evaluated the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non-oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types. We identified 369 consecutive PreservCyt samples diagnosed with ASC-US tested for HPV DNA using the LA test. The Aptima HPV test was performed on residual material in the same vial. Follow-up of 325 women was available. The gold standard used was histologically confirmed cervical intraepithelial neoplasia (CIN) grade 2+ or 3+. LA and Aptima HPV assays were positive in 44.3% and 31.7% of the cases, respectively. The concordance was 81.2%. The two tests had identical sensitivity for detecting CIN3+ [92.6% (95% CI, 75.7-99.1)] but the Aptima HPV assay showed a significantly better specificity of 73.8% (95% CI, 68.5-78.7) versus 60.1% (95% CI, 54.3-65.7) for LA for detecting CIN3+. When using CIN2+ as the gold standard the sensitivity for LA was higher than for the Aptima HPV assay [93.8% (95% CI, 82.8-98.7) versus 87.5% (95% CI, 74.8-95.3)], but the specificity was higher for the Aptima HPV assay: 78.0% (95% CI, 72.6-82.7) versus 64.3% (95% CI, 58.3-69.9). Both tests showed good and equal clinical sensitivities for detecting CIN3+, but the Aptima HPV assay had significantly higher specificity for detecting CIN2+ and CIN3+ in women aged 30 years or older with ASC-US.