BACKGROUND: Although HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with precancerous lesions of the cervix. Therefore, molecular changes in HPV 16, rather than infections per se, may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+). METHODS: The study included 75 HPV 16-positive women diagnosed with CIN 2+ or ≤CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer, and circulating concentrations of "cancer-protective" micronutrients. RESULTS: The odds of being diagnosed with CIN 2+ were 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites was ≥9.5% (OR = 0.21; 95% CI, 0.06-0.79; P = .02). CONCLUSIONS: Results suggested that CpG methylation is independently involved in the biology of HPV 16 as well as in the development of higher grades of CIN. © 2010 American Cancer Society.