Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.