Integration of human papillomavirus (HPV) DNA into the host cell genome is a frequent event in cervical carcinogenesis, even though this phenomenon does not seem to be mandatory for cervical cancer development. Our objective was to describe the load and physical state of HPV type 16 (HPV16) DNA in a series of cervical samples representative of the natural history of cervical cancer. We used a combination of three real-time PCR assays targeting E6, E2, and albumin genes to calculate HPV16 load (E6 and albumin) and the E2/E6 ratio as a surrogate of integration. This method was applied to 173 HPV16-positive cervical samples. Results show that viral load increases with the lesion grade (from 102 HPV16 DNA copies per 10(3) cells in normal samples up to 56,354 copies per 10(3) cells in cancers), while E2/E6 ratio decreases (from 1 in normal samples down to 0.36 in cancers). We propose that, according to this technique, an HPV16 viral load of higher than 22,000 copies/10(3) cells or an E2/E6 ratio of lower than 0.50 allows the identification of women with prevalent high-grade lesions or worse with a high specificity. In conclusion, both viral load and E2/E6 ratio, used in combination with an appropriate cutoff value, are suitable to screen women with prevalent cervical intraepithelial neoplasia grade 2 or 3 or cancer. Therefore, these assays would be useful in addition to routine HPV testing to more accurately identify women with (pre)cancerous lesions.