To determine the human folate requirement on the basis of changes in biochemical pathways, we studied the effect of controlled folate intakes on plasma homocysteine and lymphocyte DNA methylation and deoxynucleotide content in healthy postmenopausal women. Eight women (49-63 y of age) were housed in a metabolic unit and fed a low folate diet containing 56 microg/d of folate for 91 d. Folate intake was varied by supplementing 55-460 microg/d of folic acid (pteroylglutamic acid) to the diet to provide total folate intake periods of 5 wk at 56 microg/d, 4 wk at 111 microg/d and 3 wk at 286-516 microg/d. A subclinical folate deficiency with decreased plasma folate was created during the first two periods. This resulted in significantly elevated plasma homocysteine and urinary malondialdehyde, and lymphocyte DNA hypomethylation. The folate depletion also resulted in an increased ratio of dUTP/dTTP in mitogen-stimulated lymphocyte DNA and decreased lymphocyte NAD, changes suggesting misincorporation of uracil into DNA and increased DNA repair activity. The DNA hypomethylation was reversed with 286-516 microg/d of folate repletion, whereas the elevated homocysteine decreased with 516 but not 286 microg/d of folate. The results indicate that marginal folate deficiency may alter DNA composition and that the current RDA of 180 microg/d may not be sufficient to maintain low plasma homocysteine concentrations of some postmenopausal women.