To evaluate whether p16 staining could help to recognize underestimated cervical intraepithelial neoplasia (CIN) in women positive for high-risk human papillomavirus (HR-HPV) with negative biopsy. Out of 1,259 women undergoing a histologic study and a simultaneous HR-HPV detection using the Hybrid Capture 2 test, we selected all patients testing positive for HR-HPV and having a negative biopsy (n=139), as well as all women testing negative for HR-HPV with a biopsy of either CIN 1 (26 cases) or CIN 2 to 3 (11 cases). Of the remaining 1,083 women, we randomly selected for the purpose of controls, 50 cases negative for HR-HPV with negative biopsy and 100 cases positive for HR-HPV and with biopsy of CIN (50 CIN 1, 50 CIN 2-3). In all cases, immunohistochemical staining for p16 and a second evaluation of the initial biopsy was carried out. Thirty-four out of 139 biopsies (24.5%) testing positive for HR-HPV but having a negative biopsy were positive for p16. Thirty of these cases (21.6%) were classified as harboring a CIN (11 CIN 1, 19 CIN 2/3) after reevaluation. Both the number of cases reclassified as CIN of any grade, or as CIN 2/3, were significantly higher for cases with HR-HPV load above 100 relative light unit (P<0.005). Particular attention should be paid to biopsies from patients having positive Hybrid Capture 2. The risk of harboring undetected CIN of any type or CIN 2/3 is significantly higher for patients with high HR-HPV load. Immunostaining with p16 should be considered as a highly desirable addition to the histologic evaluation of cervical biopsy specimens in HR-HPV-positive women.