Objective. It has been suggested that colposcopy can miss a significant percentage of high-grade cervical intraepithelial neoplasia (CIN2 +). Improved disease ascertainment was evaluated by taking multiple lesion-directed biopsies. Methods. In a cross-sectional multicenter study in the Netherlands and Spain, 610 women referred to colposcopy following abnormal cervical cytology results were included. Multiple directed biopsies were collected from lesions and ranked according to impression. A non-directed biopsy of normal-appearing tissue was added if fewer than four biopsies were collected. We evaluated the additional CIN2 + yield for one and two directed biopsies. Colposcopic images were reviewed for quality control. Results. In women with at least two lesion-directed biopsies the yield for CIN2 + increased from 51.7% (95%CI; 45.7-57.7) for one directed biopsy to 60.4% (95%CI; 54.4-662, p < 0.001) for two biopsies. The highest CIN2 + yield was observed in women who were HPV16-positive, had high-grade squamous intraepithelial lesion (HSIL) cytology, and high-grade colposcopy impression. The yield increased from 83.1% (95%CI; 71.5-90.5) with one directed biopsy to 93.2% (95%CI; 83.8-97.3) with two directed biopsies. Only 4.5% additional CIN2 + were detected in biopsies not targeting abnormal areas on the cervix. Conclusions. A second lesion-directed biopsy is associated with a significant increase in CIN2 + detection. Performing a second lesion-directed biopsy and using a low threshold for abnormality of any acetowhitening should become the standard clinical practice of colposcopy. (C) 2014 Elsevier Inc. All rights reserved.