Folate intakes increased dramatically after folic acid fortification. We investigated the changes in serum folate, RBC folate, and total homocysteine (tHcy) concentrations utilizing data from National Health and Nutrition Examination Surveys (NHANES) 1988-2002. NHANES 1988-2002 were based on a stratified, multistage, probability sampling design conducted among civilian U.S. residents. The current study included 17,144, 17,213, and 11,415 measurements for serum folate, RBC folate, and tHcy, respectively. Overall, geometric mean serum folate concentrations were 149.6 and 129.8% higher in 1999-2000 and 2001-2002, respectively, than in 1988-1994 (P < 0.0001). Sex-, age-, and race-ethnicity-adjusted serum folate was significantly lower in 2001-2002 than in 1999-2000 (10.4%, P < 0.0002). The prevalence of low serum folate decreased from 18.4% in 1988-1994 to 0.8% in 1999-2000 and to 0.2% in 2001-2002 (P < 0.0001). RBC folate increased from 391 nmol/L in 1988-1994 to 618 nmol/L in 1999-2000, and to 611 nmol/L in 2001-2002. Consequently, the prevalence of low RBC folate decreased from 45.8% in 1988-1994 to 7.3% in 1999-2000 and to 7.1% in 2001-2002 (P < 0.0001). Although, RBC folate status improved after folic acid fortification in all race-ethnicities, the prevalence of low RBC folate ( approximately 20.5%) continues to be high in non-Hispanic blacks. Age-, sex-, and race-ethnicity-adjusted tHcy declined from 9.5 micromol/L in 1988-1994 to 7.6 mumol/L in 1999-2000 and to 7.9 micromol/L in 2001-2002. Although folic acid fortification contributed to significant improvement in folate status, serum folate concentrations have declined recently. This may be attributable to lower folic acid intakes.