We aimed to determine the role of HPV in the pathogenesis and outcome of oropharyngeal squamous cell carcinoma (OSCC) in lifelong nonsmoking and nondrinking patients. A case-case analysis was performed to compare the presence of HPV-DNA in tumor cells of 16 nonsmoking and nondrinking with 16 matched smoking and drinking patients (matching criteria: age at incidence, gender, tumor sublocation, tumor stage). HPV was detected using 2 PCR tests, FISH analysis, and p16INK4A immunostaining. Nonsmoking and nondrinking patients had more HPV-positive tumors than smoking and drinking patients (n=12; 75% versus n=2; 13%; P<0.001). All HPV-positive tumors showed p16INK4A overexpression, and 1 HPV-negative tumor had p16INK4A overexpression, (P<0.001). Overall survival and disease-specific survival were higher for HPV-positive compared to HPV-negative cases (P=0.027, P=0.039, resp.). In conclusion, HPV is strongly associated with OSCC of nonsmoking and nondrinking patients. Specific diagnostic and therapeutic actions should be considered for these patients to achieve a better prognosis.