Primary human papillomavirus (HPV)-based screening results in a 2-5% lower specificity for cervical intraepithelial neoplasia Grade 2 or worse (CIN21) compared to Pap cytology. To identify HPV-positive women with CIN21, we retrospectively evaluated the cross-sectional and longitudinal performance of p16/Ki-67 dual-stained cytology in HPV-positive women with normal cytology participating in population-based cervical screening. Conventional Pap cytology specimens of 847 of these women derived from the VUSA-Screen study were dual-stained for p16/Ki-67. Cross-sectional clinical performance in detecting CIN3 or worse (CIN31), and CIN21 was compared to that of baseline HPV genotyping. Moreover, 5-year cumulative incidence risks (CIR) for CIN31 (CIN21) were determined. The sensitivity of p16/Ki-67 dual-stained cytology for CIN31 (CIN21) was 73.3% (68.8%) with a specificity of 70.0% (72.8%). HPV16/18 genotyping showed a sensitivity for CIN31 (CIN21) of 46.7% (43.8%), with a specificity of 78.3% (79.4%). The 5-year CIR for CIN31 in HPV-positive women with normal cytology was 6.9%. Testing these women with p16/Ki-67 dual-stained cytology resulted in a significantly lower CIN31 5-year CIR of 3.3% (p 5 0.017) in case of a negative test result. A negative HPV16/18 genotyping test result also led to a lower 5-year CIN31 CIR of 3.6%. p16/ Ki-67 dual-stained cytology detects more than 70% of underlying CIN31 lesions in HPV-positive women with normal cytology at baseline and is therefore suitable for triaging these women to colposcopy. Furthermore, the CIN31 5-year CIR of 3.3% after a negative dual-stain result is significantly lower compared to the 5-year CIR of 6.9% in women without p16/Ki-67 dualstained cytology triage.Persistent infections with carcinogenic human papillomavirus (HPV) genotypes represent the main causative event in the multistep process of cervical carcinogenesis. Recent efforts to improve cervical screening have focused on introducing HPV testing as a conjunct to Pap cytology testing, or as a primary screening tool. Unlike cytology, HPV testing is objective and has consistently been shown to be more sensitive for the detection of cervical intraepithelial neoplasia Grade 2 (Grade 3) or worse (CIN21/31) compared to cytology-based testing(for CIN31 94% vs. 65%).1,2 Based on this evidence, Key words: p16/Ki-67 dual-stained cytology, cervical cytology, human papillomavirus, genotyping Abbreviations: BMD: borderline mild dyskaryosis; CIN21: cervical intraepithelial neoplasia Grade 2 or worse; CIN31: cervical intraepithelial neoplasia Grade 3 or worse; CIR: cumulative incidence risks; EIA: enzyme immunoassay; HC2: hybrid capture 2; HPV: human papillomavirus; LBC: liquid-based cytology; NPV: negative predictive value; PPV: positive predictive value;