To investigate the effects of pregnancy on folate metabolism, we conducted an 84-d study in second-trimester (gestational wk 14-25) pregnant women (n = 6) and nonpregnant controls (n = 6) with stable-isotopic tracer methods. All subjects were fed a diet containing approximately 272 nmol/d (120 microg/d) folate from food, along with supplemental folic acid that contained 15% [3',5'-(2)H(2)] folic acid ([(2)H(2)]folic acid) during d 1--41 and that was unlabeled during d 42--84 to yield a constant total folate intake of 1.02 or 1.93 micromol/d (450 or 850 microg/d). Isotopic enrichment of plasma folate, urinary folate and the urinary folate catabolites para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) was determined at intervals throughout the study. The labeling of pABG and ApABG reflected that of tissue folate pools from which the catabolites originate. After the intake of labeled folic acid was terminated on d 41, labeling of urinary folate exhibited a biphasic exponential decline with distinct fast and slow components. In contrast, during d 42--84, the enrichment of urinary pABG and ApABG exhibited primarily monophasic exponential decline, and plasma folate underwent little decline of labeling during this period. Pregnant women and controls did not differ in estimates of body folate pool size and most aspects of the excretion of labeled urinary folate and catabolites, rates of decline of excretion, and areas under the curves for folate and catabolite excretion. Pregnant women, however, tended to have a slower rate of decline of pABG than ApABG and higher enrichment at d 42 of ApABG and pABG. These data support and extend our previous findings indicating that pregnancy (gestational wk 14--26) causes subtle changes in folate metabolism but does not elicit substantial increases in the rate or extent of folate turnover at these moderately high folate intakes.