Human papillomavirus (HPV) is a circular double-stranded DNA virus implicated in at least 90% of cervical and anogenital cancers and has been observed in approximately 20% of squamous cell carcinomas of the head and neck (SCCHN). Transcription of the viral oncogenes E6 and E7 is regulated by expression of the E2 protein. Disruption of the E2 gene sequence due to viral integration results in upregulation of E6 and E7, which promote tumorigenesis by abrogating p53 and pRb functions. HPV integration sites in cervical and anogenital cancers have been mapped primarily to chromosomal fragile sites and in some cases have been shown to integrate within tumor suppressor genes or other cancer-related genes. To study viral integration sites in SCCHN, we examined an HPV16-infected SCCHN cell line cultured from a tongue-base tumor. HPV fluorescence in situ hybridization (FISH) revealed multiple integrated viral DNA copies in blocks throughout the genome. Sequential FISH and spectral karyotyping identified integration sites on chromosomes 3, 6, 9q, 13q and t(1;8)(q;?). Restriction site-polymerase chain reaction (RS-PCR) was performed to identify the viral-cellular junctions. Sequence analyses confirmed integration sites at 9q31.1 and 6p21 and revealed a novel junction at 16p12.3. Subsequent chromosome breakage studies suggested that the observed viral-cellular integration sites may have occurred within common fragile sites. Additional studies using RT-PCR for E6--E7 viral transcripts showed oncoprotein expression from episomal and integrated viral sequences. Our results suggest that viral integration of HPV in SCCHN appears to occur nonrandomly through targeting specific chromosomal sequences prone to breakage.