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 1.0.8% (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.