Human papillomaviruses (HPV) are associated with a subset of head and neck squamous cell carcinoma (HNSCC), particularly HPV16. This study analyzed the presence and genotype of high risk HPVs, viral DNA load and transcription of the E6/E7 mRNAs, in 231 consecutive HNSCC. Twelve out of 30 HPV16 DNA-positive tumors displayed high E6/E7 mRNAs levels and were localized in the oropharyngeal region. While HPV-free and non-transcriptionally active HPV-related patients showed similar 5-years survival rates, E6/E7 expression was associated with a better prognosis. This emphasizes the importance of considering the transcriptional status of HPV-positive tumors for patient stratification. A gene expression profiling analysis of these different types of tumors was carried out. The most significant differentially expressed gene was CDKN2A, a known biomarker for HPV-related cancer. Assessing both the expression level of the E6/E7 mRNAs and of CDKN2A in HNSCC is required to detect active HPV infection. Chromosomic alterations were investigated by Comparative Genomic Hybridation (CGH) analysis of tumors with transcriptionally active HPV and HPV-negative tumors. The loss of the chromosomal region 16q was found to be a major genetic event in HPV-positive lesions. A cluster of genes located in 16q21-24 displayed decreased expression levels, notably APP-BP1 that is involved in the modulation of the transcriptional activity of p53. In conclusion, this study highlights important criteria required to predict clinically active HPV infection, identifies new biological pathways implicated in HPV tumorigenesis and increases the understanding of HPV-HNSCC physiopathology that is required to develop new targets for therapy.