Infection with human papillomavirus (HPV) type 16 accounts for about half of cervical cancers worldwide. This study investigated the seroepidemiology of HPV-16 infection in the United States by using a population-based survey. Serum samples and questionnaire data were collected from 1991 to 1994 for the National Health and Nutrition Examination Surveys. HPV-16-specific IgG antibody was detected by use of an HPV-16 virus-like particle ELISA. HPV-16 seropositivity in the US population aged 12-59 years was 13.0% (95% confidence interval, 11.5%-14.7%). Seroprevalence was higher in women (17.9%) than in men (7.9%). Age, race/ethnicity, and number of lifetime sex partners were associated with HPV seropositivity in women. Race/ethnicity, age at first intercourse, urban/nonurban residence, years of sexual activity, and having had sex with a man were associated with HPV seropositivity in men. Information on HPV-16 seroepidemiology will be important for designing prevention efforts including vaccine programs.