Detection of E6/E7 mRNA expression using the real-time nucleic acid sequence-based amplification assay (NASBA) PreTect HPV-Proofer was compared with results of human papillomavirus (HPV) DNA detection in 98 paraffin-embedded samples from patients with cervical adenocarcinoma. HR-HPV DNA was detected in 61 (62%), while HR-HPV E6/E7 mRNA was detected in 63 (64%) of the samples. Correlation between results from DNA analyses and the E6/E7 mRNA assay showed consistent results in 87% of samples (47 of 54). The results from these two methods in detecting presence of HPV infection of any type agreed in 77%. Overall agreement between the methods was seen in 82 of the 98 cases (84%). When evaluating change in sensitivity for detection of HPV positives by adding more HPV types to the HPV DNA assay, maximum sensitivity was reached by targeting four HPV types. The coverage of HPV DNA presence was 76.9%, while the E6/E7 mRNA assay achieved a maximum coverage of 80.8% using only three HPV types. Thus, E6/E7 oncogene expression analysis may provide a more objective test for assessment of neoplastic glandular cells. Further studies may reveal whether the clinical performance of the E6/E7 mRNA assay will be of prognostic value in management of cervical adenocarcinoma.