OBJECTIVE: To estimate the optimal relative light unit ratio, and correspondingly viral load, of the hybrid capture II oncogenic human papillomavirus deoxyribonucleic acid test for detecting cervical intraepithelial neoplasia (CIN). METHODS: Women with abnormal cytology were referred for colposcopy, and a cervical swab or brush specimen was obtained for human papillomavirus testing. Sensitivities, specificities, and likelihood ratios of different relative light unit ratio cutoffs were calculated using a reference standard of colposcopy or biopsy of either CIN II+ (CIN II, III, or carcinoma), or CIN I+ (CIN I, CIN II+). The receiver operating characteristic curve was used to estimate optimal test-positive cutoff points for the hybrid capture II test. RESULTS: CIN II+ was found in 18.7% (98 of 524) and CIN I in 10.5% (55 of 524) of the women. The optimal relative light unit ratio was 15.56, giving a sensitivity and specificity of 82.7% and 73.2% for CIN II+, and 74.2% and 77.8% for CIN I+. In a stratified analysis, a higher relative light unit cutoff (15.19) optimized sensitivity and specificity for CIN II+ (sensitivity 81.8%, specificity 51.5%) for women with low-grade squamous intraepithelial lesions cytology, whereas the optimal cutoff was 2.36 (sensitivity 100%, specificity 73.0%) for women with atypical squamous cells of undetermined significance, yielding referral rates of 53.3% and 28.7%, respectively. Use of a swab or brush was not associated with the level of human papillomavirus detected (P > .05). CONCLUSION: Use of a higher cutoff for the relative light unit ratio (higher viral load) of the hybrid capture II test may improve the management of women, especially those with low-grade squamous intraepithelial lesions cytology.