HPV infection has long been implicated in the development of cervical carcinoma. There is strong evidence for association of high-risk HPV types 16 and 18 with cervical intraepithelial neoplasia (CIN) grades 2 and 3, and integration of viral DNA of these types into the host genome has been suggested to play an important role in progression to invasive cervical carcinoma. However, the existing techniques for detection of integrated DNA in clinical specimens are time-consuming and require large amounts of template DNA, often unavailable for small premalignant CIN lesions. In this study, a novel, two stage PCR assay was designed to discriminate between integrated and episomal HPV 16 DNA. The first stage was designed to determine whether intact HPV genomes were present. This initial PCR analysis of the entire viral genome in eight segments was successfully applied to authentic human cervical cancers. The second stage consisted of an ANCHOR-PCR-based analysis, developed specifically to discriminate between integrated and episomal HPV DNA, that was successfully tested with cloned HPV containing plasmids used to mimic both episomal and integrated viral DNA. Further optimization and validation will be required for application of the second stage to clinical specimens. The entire assay was developed to be applicable to small colposcopic biopsies or cervical scrape samples, representative of those acquired in routine clinical investigation of CIN 2 or CIN 3, in which determination of the physical state of HPV DNA may provide prognostically valuable information.