We have previously shown that women with a high titer of human papillomavirus type 16 (HPV16) in cervical epithelial cells have an increased risk of developing cervical carcinoma in situ. In order to study the relationship between viral DNA amount and risk of cervical carcinoma for the HPV types most commonly found in cervical tumors, we developed a real-time PCR assay for the detection and quantification of HPV16, -18, -31, -33, -35, -39, -45, -52, -58, and -67. These HPV types are analyzed in two reaction tubes, allowing for independent quantification of three viral types, or groups of viral types, in each reaction. A separate reaction is used for estimating the number of a nuclear single-copy gene and is used to calculate the HPV copy number per genomic DNA equivalent in the sample. The system has a dynamic range from 10 2 to 10 7 HPV copies per assay and is applicable to both fresh clinical samples and DNA extracted from archival samples. Reconstitution experiments, made to mimic infections with several HPV types, shows that individual HPV types can be detected in a mixture as long as they represent 1 to 10% of the main type. The system was evaluated with respect to technical specificity and sensitivity, reproducibility, reagent stability, and sample preparation protocol and then used to analyze clinical samples. This homogeneous assay provides a fast and sensitive way for estimating the viral load of a series of the most frequent oncogenic HPV types in biopsies, as well as cervical smear samples.Cervical carcinoma is considered to be the third most common cancer in women in the world. In 1994 an estimated 55,000 women in the United States were diagnosed with carcinoma in situ of the cervix, with an additional 15,000 cases of invasive cancer (15). Although in the United States and Europe major progress has been made in the control of cervical cancer, it remains a significant cause of morbidity and mortality in the developing world.Infection by certain types of human papillomavirus (HPV) is the single most important risk factor for the development of cervical cancer (10, 17). More than 99% of cervical cancer biopsies have been found to contain HPV DNA, most commonly HPV type 16 (HPV16), followed by HPV18, -45, -31, and -33 (1, 22). Given the importance of HPV infection in the etiology of cervical cancer, a large number of methods have been developed for detecting of the virus or for identifying the cellular changes resulting from viral transformation (8).We have previously described an assay based on real-time PCR for the detection and quantification of high-risk HPV DNA (7). The 5Ј exonuclease assay, employed in Taqman, is based on the ability of the 5Ј-to-3Ј exonuclease activity of Taq polymerase to cleave a dually labeled, nonextendable hybridization probe during the extension phase of the PCR (4, 5, 11, 13). Other groups have applied the 5Ј exonuclease assay either for endpoint determination of the amount of HPV PCR product (18) or for real-time detection of HPV (19-21). Additional methods for the quantif...