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The elevated risk for incident head and neck cancer among human papillomavirus (HPV)-16-seropositive individuals has substantiated a role for HPV in the etiology of head and neck cancers. The relationship between HPV seroreactivity and prevalent oral HPV infection in men and women without cancer has yet to be investigated.The goal of this study was to evaluate a possible association between oral HPV infection and HPV seroreactivity after adjustment for gender, sexual behaviors, and sexually transmitted disease.A cross-sectional study of factors associated with HPV-16, −18, and −33 seroreactivity was performed in a population of 586 men and women with and without HIV infection. Antibodies in sera were measured by use of a virus-like protein (VLP)-based enzyme-linked immunosorbent assay. Exfoliated cells from the tonsillar and oral mucosa were analyzed for the presence of 38 mucosal HPV types by polymerase chain reaction.Women had significantly greater seroreactivity for all HPV types investigated when compared with men (odds ratio, 4.3; 95% confidence interval, 3.0–6.0). Seroprevalence was greatest in men and women aged 35 to 45 years. Tonsillar HPV infection, oral sex with men, and HIV infection were independently associated with HPV seroreactivity in men after adjustment for age and number of sexual partners. In women, HSV-2 seropositivity and a history of sexually transmitted diseases were similarly important. Oral and tonsillar HPV infection were not associated with HPV seroreactivity in women.HPV seropositivity is associated with sexually transmitted diseases among women and possibly mucosal HPV exposures in men. Tonsillar HPV infection could impact seroprevalence, particularly in men.