BACKGROUND. Congenital cytomegalovirus (CMV) infection causes permanent disabilities in more than 5500 children each year in the United States. The likelihood of congenital infection and disability is highest for infants whose mothers were CMV seronegative before conception and who acquire infection during pregnancy. METHODS. To provide a current, nationally representative estimate of the seroprevalence of CMV in the United States and to investigate trends in CMV infection, serum samples from the National Health and Nutrition Examination Survey (NHANES) 1999-2004 were tested for CMV-specific immunoglobulin G antibody, and results were compared with those from NHANES III (1988-1994). Individuals aged 6-49 years (21,639 for NHANES III and 15,310 for NHANES 1999-2004) were included. RESULTS. For NHANES 1999-2004, the overall age-adjusted CMV seroprevalence was 50.4%. CMV seroprevalence was higher among non-Hispanic black and Mexican American children compared with non-Hispanic white children and increased more quickly in subsequent age groups. CMV seropositivity was independently associated with older age, female sex, foreign birthplace, low household income, high household crowding, and low household education. Compared with NHANES 1988-1994, the overall age-adjusted CMV seroprevalence for NHANES 1999-2004 was not significantly different. CONCLUSIONS. Many women of reproductive age in the United States are still at risk of primary CMV infection during pregnancy. There is an urgent need for vaccine development and other interventions to prevent and treat congenital CMV. The substantial disparities in CMV risk among seronegative women suggest that prevention strategies should include an emphasis on reaching racial or ethnic minorities and women of low socioeconomic status.