Objectives: To assess the effects of supplementation with the diastereoisomer of 5-methyltetrahydrofolate ([6S]5-methylTHF), as an alternative supplement for folic acid, on folate absorption and elimination, in two age groups. Design: A randomized, double-blind intervention study. Subjects: A total of 12 young (<30 y) and 12 middle-aged (≥50 y) healthy volunteers were recruited. Methods: Volunteers were randomized to receive daily supplementation with 400 μg folic acid or equimolar amounts of [6S]5-methylTHF during 5 weeks. Before and after supplementation, absorption and initial elimination were calculated following oral [2H2]folic acid test doses using isotope kinetics in plasma. Results: Folic acid absorption was lower in the middle-aged as compared to the young adults, both before (P=0.03) and after (P=0.05) supplementation. In the young adults, absorption decreased by 22% after [6S]5-methylTHF and increased by 21% after folic acid (P=0.02). In the other age group, no such changes were found. The folate rate constant of elimination increased after folic acid supplementation in the young (+50%; P=0.05) but not in the middle-aged (+18%; P=0.5) adults. Conclusions: Young adults show increased folate turnover after folic acid supplementation relative to the effect of [6S]5-methylTHF supplementation. Similar differences are not observed in middle-aged adults, in whom folic acid absorption was found to be lower as compared to the young adults.