Fresh-frozen biopsies were obtained from 61 patients at diagnosis of squamous cell carcinoma of the head and neck (HNSCC) for study of the prevalence and physical status of human papillomavirus (HPV) DNA. The frequency of HPV DNA and genotypes were determined by SPF10 PCR screening with a general probe hybridization and INNO-LiPA HPV genotyping assay. In addition, a single-phase PCR with primers FAP 59/64 and a nested PCR with primers CP 65/70 and CP 66/69 served to detect particularly cutaneous HPV types. By the sensitive SPF10 PCR and INNO-LiPA assay, 37 of 61 (61%) samples were positive for HPV. HPV-16 was the most frequently detected type (31 of 37, 84%). Multiple infections were found in 8 of 37 (22%) of the HPV-positive samples, and co-infection by HPV-16 and HPV-33 was predominant. No cutaneous HPV types were detected. Patients with HPV-positive tumors had similar prognosis as those with HPV-negative ones. Real-time PCR analysis of the HPV-16 positive samples indicated the presence of integrated (11 of 23, 48%), episomal (8 of 23, 35%) and mixed forms (4 of 23, 17%) of HPV DNA. The viral load of HPV DNA exhibited large variation. The median copy numbers of E6 DNA in tonsillar specimens were approximately 80,000 times higher than that in nontonsillar HNSCC ones. Patients with episomal viral DNA were more frequently found to have large (T3–T4) tumors at diagnosis than were those with integrated or mixed forms. © 2003 Wiley-Liss, Inc.