Choline and folate share methylation pathways and, in studies of rats, were shown to be metabolically inter-related. To determine whether choline status is related to folate intake in humans, we measured the effect of controlled folate depletion and repletion on the plasma choline and phosphatidylcholine concentrations of 11 healthy men (33-46 y) and 10 healthy women (49-63 y) fed low-choline diets in two separate metabolic unit studies. Total folate intake was varied by supplementing low folate (25 and 56 microg/d for men and women, respectively) and low choline (238 and 147 mg/d for men and women, respectively) diets with pteroylglutamic acid for 2-6 wk following folate-depletion periods of 4-5 wk. The low folate/choline intakes resulted in subclinical folate deficiencies; mean plasma choline decreases of 28 and 25% in the men and women, respectively; and a plasma phosphatidylcholine decrease of 26% in the men (P < 0. 05). No functional choline deficiency occurred, as measured by serum transaminase and lipid concentrations. The decreases in choline status measures returned to baseline or higher upon moderate folate repletion and were more responsive to folate repletion than plasma folate and homocysteine. Feeding methionine supplements to the men did not prevent plasma choline depletion, indicating that folate is a more limiting nutrient for these methylation pathways. The results indicate that 1) choline is utilized as a methyl donor when folate intake is low, 2) the de novo synthesis of phosphatidylcholine is insufficient to maintain choline status when intakes of folate and choline are low, and 3) dietary choline is required by adults in an amount > 250 mg/d to maintain plasma choline and phosphatidylcholine when folate intake is low.