The human papillomavirus (HPV) has been implicated as an etiological factor in a subset of head and neck squamous cell carcinoma (HNSCC). Because circulating tumor DNA has previously been detected in the sera of patients with advanced HNSCC (stage III or IV), we hypothesized that HPV DNA might be present in the sera of HPV-positive HNSCC patients. Serum DNA extracts from 70 patients with HNSCC were screened for HPV using conventional PCR and a real-time quantitative assay. All samples subjected to conventional PCR were further tested by dot blot hybridization, and positives were confirmed by Southern blotting. Paired tumor DNA from archived tissues was then similarly screened for HPV genomic material (n = 51) or tested by in situ hybridization (n = 19). HPV-16 DNA was detected with L1 primers in 0 of 65 sera and in 15 of 70 (21%) tumors. Conventional PCR with E7 primers and Southern blot hybridization detected HPV-16 DNA in four (6%) sera. Using real-time quantitative PCR, six samples were found to contain various levels of circulating HPV DNA (mean, 12 copies/ml; range, <1-35.) All six serum-positive patients had corresponding tumors positive for E7. Four of these patients with HPV-positive tumors later developed distant metastases, suggesting that HPV DNA in serum may represent occult hematogenous spread of cancer cells in this subset of patients. Although a much larger prospective trial is required, the presence of HPV genomic material in serum DNA of HPV-positive HNSCC patients may serve as a useful marker of early metastatic disease.