Elevated circulating total homocysteine (tHcy) concentrations are related to increased risk for cardiovascular disease. Folic acid fortification resulted in a significant decrease in circulating tHcy in the United States. In the post-folic acid fortification period, associations between plasma tHcy and demographic, health, and lifestyle factors and B vitamin status were investigated. Data from the 3 recent NHANES cycles (1999-2004) were used to study the associations among plasma tHcy and sex, race/ethnicity, age, BMI, blood pressure (BP), serum creatinine, serum cotinine, vitamin/mineral supplement use, and folate and cobalamin status in 16,254 persons (8329 men, 7925 women). Multivariate-adjusted plasma tHcy was approximately 9.7% higher in men than in women (P < 0.0001), approximately 5.3% higher in non-Hispanic whites than in non-Hispanic blacks (P < 0.0001), approximately 64.5% higher in individuals aged >or=60 y than in individuals aged <20 y (P < 0.0001), and approximately 5% higher in supplement nonusers than in supplement users (P < 0.0001). Persons in the 4th quartile for serum creatinine, serum cotinine, systolic BP, and serum methylmalonic acid (MMA) had approximately 36.1, approximately 13.4, approximately 5.6, and approximately 16.2%, respectively, higher tHcy than those in the 1st quartile (P < 0.0001). Plasma tHcy was inversely associated with serum folate (P < 0.0001), RBC folate (P < 0.0001), and serum cobalamin (P < 0.0001). In the post-folic acid fortification period, sex, race/ethnicity, age, systolic BP, supplement use, RBC folate, and serum creatinine, cotinine, folate, and cobalamin are significant determinants of plasma tHcy. Serum MMA was strongly associated with plasma tHcy.