Incidence of oropharyngeal squamous cell carcinoma (OSCC) increased 3% annually from 1973 to 2001. OSCC's can be attributed to tobacco and alcohol, but 25% are unlinked to typical risks. Case-control studies on HPV detection in non-smoking/non-drinking (NS/ND) OSCC patients have not previously been performed. The primary objective of this study was to determine whether high-risk HPV infection was significantly associated with development of oral squamous malignancy in non-smokers/non-drinkers. A chart review of 802 OSCC patients from the UNC Pathology Archives (1995-2006) yielded 40 non-smoker/non-drinker subjects. Utilizing a case-control design, 18 cancer cases and 22 benign biopsy controls were consecutively identified. Biopsy tissue was subjected to (i) HPV-L1 consensus PCR and sequencing (ii) real-time PCR. Chi-square and logistic regression analysis was employed. Logistic regression analysis determined that cases were 6.1 (OR 95% CI, 1.3-28) times more likely to have HPV infection in their tumors than controls. High-risk HPV-DNA was readily detected in the tonsils and base of tongue (oropharynx) of 14/18 cases and 6/22 controls by both consensus and real-time PCR. Of high-risk HPV containing lesions, 85% (17/20) originated in the oropharynx (chi-square, p=0.03). High risk HPV was also detected in benign biopsies of the oropharynx in 30% (3/10) of individuals who had a previous oral cancer (chi-square, p=0.006). The infectious nature of OSCC in NS/ND was revealed by consistent detection of HPV, suggesting HPV's potential role in transforming oral epithelium, providing further evidence of the need to screen the oropharynx for HPV in NS/ND.