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Toxoplasma gondii can cause congenital, neurologic, ocular, and mild or asymptomatic infection. To determine the U.S. prevalence of T. gondii infection, we tested sera collected from the National Health and Nutrition Examination Survey (NHANES) 1999–2004 for T. gondii immunoglobulin G antibodies in persons 6–49 years old and contrasted the results to those comparable in NHANES III (1988–1994) (ages 12–49 years). Of the 17,672 persons examined in NHANES 1999–2004, 15,960 (90%) were tested. The age-adjusted T. gondii seroprevalence among persons 6–49 years old was 10.8% (95% confidence limits [CL] 9.6%, 11.9%), and among women 15–44 years old, 11.0% (95% CL 9.5%, 12.4%). T. gondii seroprevalence declined from 14.1% to 9.0% (P < 0.001) from NHANES III to NHANES 1999–2004 among U.S.-born persons ages 12–49 years. Although T. gondii infects many persons in the U.S., the prevalence has declined in the past decade.