We examined the relationship between serum folate and total homocyst(e)ine levels by determining protein-bound homocyst(e)ine in stored serum from 19 subjects with subnormal serum folate (less than 2 ng/mL), 137 subjects with low normal serum folate (between 2.0 and 3.9 ng/mL), 44 subjects with normal serum folate (between 4.0 and 17.9 ng/mL), and 38 subjects with high serum folate (above 18 ng/mL). Eighty-four percent of the subjects with subnormal serum folate and 56% of the subjects with low normal serum folate had more than 7.05 nmol/mL serum total homocyst(e)ine (ie, more than two standard deviations above the normal mean). Thirty-two percent of these subjects had more than a three-fold increase in serum total homocyst(e)ine. These observations support the hypothesis that depletion of tissue folate causes homocysteinemia in nonhomocystinuric subjects. Subnormal as well as low normal concentrations of serum folate appear to produce an accumulation of homocyst(e)ine. In addition, relatively normal levels of serum total homocyst(e)ine were observed in four pregnant women with low serum folate, supporting previous suggestions of an influence of female sex hormone(s) in homocysteine metabolism.