BACKGROUND. The objective of this study was to assess the rate and possible reasons for false-negative (FN) reflex human papillomavirus (HPV)-DNA tests. METHODS. The authors reviewed all ThinPrep cervical specimens that were submitted for reflex HPV-DNA testing using the Digene Hybrid Capture II (HC2) method from January 2002 to January 2004. Follow-up biopsies were reviewed. The results were considered HPV-FN if the HPV-DNA test was negative and the biopsy was positive for grade ≥2 cervical intraepithelial neoplasia (CIN2+), and the results were considered true positive (HPV-TP) if the HPV-DNA test was positive and the biopsy showed CIN2+. HPV-FN cases were compared with HPV-TP cases regarding the grade and extent of CIN, the number of abnormal cells on the original ThinPrep slide, and the presence of amplifiable, viral DNA on biopsy. RESULTS. In total, 1520 (66%) of 2309 patients who had diagnoses of atypical squamous cells of undetermined significance (ASCUS) were negative for HPV DNA and 789 patients of 2309 patients (34%) were positive for HPV DNA. Three hundred sixteen women (40%) who had a positive HPV-DNA test underwent a biopsy. Of those, 36 biopsies (11%) showed CIN2+ (HPV-TP), and 154 biopsies (66%) showed CIN1. Cervical tissue was available for review from 82 women who had negative HPV-DNA tests; of these, 6 tissue samples (7%) showed CIN2+ (HPV-FN), and 13 tissue samples (16%) showed CIN1. Therefore, in the total ASCUS population that was triaged with reflex HPV testing, there were at least 42 women who were diagnosed with CIN2+, for an estimated CIN2+ FN fraction of 14% (6 of 42 women). HPV-FN lesions were smaller (but the difference was not statistically significant) and shed significantly fewer abnormal cells than HPV-TP cases. Polymerase chain reaction testing for viral DNA in the biopsy was detected in 3 of 6 women who had HPV-FN results; none of those positive results demonstrated a viral type that was not included in the Digene probes. CONCLUSIONS. Although the rate of FN high-grade lesions was significantly higher than that reported in the ASCUS/Low-grade Squamous Intraepithelial Lesion Triage trial, most missed lesions were small and shed few abnormal cells. It was assumed that those lesions were either in early stages or in regressing stages, which made their clinical significance uncertain. © 2007 American Cancer Society.