The purpose of this study was to evaluate the clinical use of the Hybrid Capture (HC)-II system for the detection of human papillomavirus (HPV) DNA to identify women at risk of progression to high grade squamous intraepithelial lesions (HGSIL) and carcinomas by differentiating low risk (LR) HPV types (6, 11, 42, 43, 44) and high/intermediate risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Five hundred and ninety-six women were enrolled in the study. Among them, 466 attended the hospital for routine cytologic screening and 130 were referred for colposcopy because of an abnormal Pap smear. The presence of HPV DNA was tested in cervical samples collected with the Digene Cervical Sampler in Digene Specimen Transport Medium (Digene Corporation, Silver Spring, MD, U.S.A.) using the HC-II assay. Results were compared with those obtained by polymerase chain reaction (PCR) using the MY09-MY11 primers followed by several hybridizations with specific probes. The overall HPV positivity was 32.9% by HC-II and 37.8% by PCR. Among cytologically normal smears, 19.5% were positive by HC-II (14.3% HR) and 25.1% by PCR. Of the atypical squamous cells of undetermined significance samples, 52.9% were positive by HC-II (41.1% HR) and 55.9% by PCR. Of the low grade SIL, 64.5% were positive by HC-II (59.4% HR) and 68.7% by PCR. The HPV positivity rate was found identical by both techniques in high grade smears (81.6%) and squamous cervical carcinomas (100%). By using PCR as the reference method, the sensitivity of HC-II was higher among women with abnormal cytology than with normal cytology (87.3% vs. 70%). Specificity was 80.8% and 97.5%, respectively. In summary, these results indicate that the HC-II method and MY-PCR identified nearly equivalent prevalences of HPV in cervical smear specimens.