High‐risk human papillomaviruses (HPVs) have been proposed to be associated with a subset of head and neck cancers (HNSCCs). However, clear biological evidence linking HPV‐mediated oncogenesis to the development of HNSCC is hardly available. An important biological mechanism underlying HPV‐mediated carcinogenesis is the inactivation of p53 by the HPV E6 oncoprotein. In the present study we investigated this biological relationship between HPV and HNSCC. In total 84 HNSCC tumors were analyzed for the presence of high‐risk HPV nucleic acids by DNA polymerase chain reaction‐enzyme immunoassay (PCR‐EIA) and E6 reverse transcriptase (RT)‐PCR as well as for the presence of mutations in the p53 gene. We found 20/84 HPV16 DNA‐positive cases with one or more DNA assays, 10 of which were consistently positive with all assays. Only 9/20 cases showed E6 mRNA expression, indicative for viral activity. Only these nine E6 mRNA‐positive cases all lacked a p53 mutation, whereas both the other HPV DNA‐positive and HPV‐DNA negative tumors showed p53 mutations in 36% and 63% of the cases, respectively. Moreover, only in lymph node metastases of HPV E6 mRNA‐positive tumors both viral DNA and E6 mRNA were present. Our study provides strong biological evidence for a plausible etiological role of high‐risk HPV in a subgroup of HNSCC. Analysis of E6 mRNA expression by RT‐PCR or alternatively, semiquantitative analyses of the viral load, seem more reliable assays to assess HPV involvement in HNSCC than the very sensitive DNA PCR analyses used routinely. © 2001 Wiley‐Liss, Inc.