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Evaluation of sexual behaviors is essential to better understand the epidemiology of sexually transmitted infections and their sequelae.The National Health and Nutrition Examination Surveys (NHANES) is an ongoing probability sample survey of the US population. Using NHANES sexual behavior data from 1999 to 2012, we performed the following: (1) trend analyses among adults aged 25 to 59 years by 10-year birth cohorts and (2) descriptive analyses among participants aged 14 to 24 years. Sex was defined as vaginal, anal, or oral sex.Among adults aged 25 to 59 years, median age at sexual initiation decreased between the 1940–1949 and 1980–1989 cohorts from 17.9 to 16.2 among females (Ptrend < 0.001) and from 17.1 to 16.1 among males (Ptrend < 0.001). Median lifetime partners increased between the 1940–1949 and 1970–1979 cohorts, from 2.6 to 5.3 among females (Ptrend < 0.001) and from 6.7 to 8.8 among males (Ptrend < 0.001). The percentage of females reporting ever having a same-sex partner increased from 5.2% to 9.3% between the 1940–1949 and 1970–1979 cohorts (Ptrend < 0.001). Among participants aged 14 to 24 years, the percentage having had sex increased with age, from 12.5% among females and 13.1% among males at age 14 years to more than 75% at age 19 years for both sexes. Among sexually experienced 14- to 19-year-olds, 45.2% of females and 55.0% of males had at least 3 lifetime partners; 39.4% of females and 48.6% of males had at least 2 partners in the past year. The proportion of females aged 20 to 24 years who reported ever having a same-sex partner was 14.9%. The proportion of participants aged 14–19 or 20–24 years reporting ever having sex did not differ by survey year from 1999 to 2012 for either males or females.Sexual behaviors changed with successive birth cohorts, with more pronounced changes among females. A substantial proportion of adolescents are sexually active and have multiple partners. These data reinforce existing recommendations for sexual health education and sexually transmitted infection prevention targeting adolescents before sexual debut.