The clinical utility of HPV 16 and 18 viral loads remains debated. The aim of this study was to assess the clinical significance of HPV 16 and 18 viral load and to determine a cut-off for optimal prediction of grade 2 or higher cervical intraepithelial neoplasia among patients referred to colposcopy. A total of 186 cervico-vaginal specimens harboring HPV 16 and/or 18 obtained at the time of colposcopy from patients without previous cervical neoplasia were tested for HPV 16 and 18 detection and quantitation using quantitative duplex real-time PCR method. Grade 2 or higher cervical intraepithelial neoplasia was diagnosed in 87 (46.8%) cases. Only HPV 16 median viral load increased significantly with the lesion grade: 9.1 × 104 in normal cervix or grade 1 cervical intraepithelial lesion versus 4.0 × 106 copies per million cells in grade 2 or higher cervical intraepithelial lesion (P < 0.001). The highest predictive value for grade 2 or higher cervical intraepithelial lesion was observed with a HPV 16 viral load cut-off of 3.0 × 106 copies per million cells (91% specificity, 58.2% sensitivity). Using this cut-off, the highest predictive value of HPV 16 viral load was observed among those referred for previous low-grade abnormal cervical cytology (96.4% specificity, 88% sensitivity). HPV 18 quantitation showed very poor predictive value. Specific attention should be given when performing colposcopic examination of women with an HPV 16 viral load higher than 3.0 × 106 copies per million cells, especially among those referred after a low-grade abnormal cytology. J. Med. Virol. 84:306–313, 2012. © 2011 Wiley Periodicals, Inc.