Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV⁺ HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV⁻ HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV⁺ HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV⁻ HNC. Moreover, HPV⁺ cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV⁺ HNC and HPV⁻ HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV⁺ and HPV⁻ cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV⁺ cancers. [Cancer Res 2007;67(10):4605–19]