During intestinal absorption, dietary polyglutamyl folates are hydrolyzed to monoglutamyl folates by pteroylpolyglutamate hydrolase (folate conjugase). This enzyme is present in the brush border and intracellular fractions of human jejunal mucosa. We compared the activities of brush border and intracellular folate conjugase (BBFC and ICFC), and other mucosal enzymes in the jejunum in continuity and bypassed loop in nine patients who underwent intestinal reconstitution operations 3 to 9 yr after jejunoileal bypass. Control jejunum was obtained from seven obese patients undergoing gastric bypass surgery. Jejunal morphometry showed mucosal hyperplasia with taller villi, larger crypts, and no change in cell size in the jejunum in continuity as compared to control or bypass jejunum. In brush border fractions, specific activities of sucrase and BBFC were significantly greater in the jejunum in continuity than in the control or bypass jejunum. In contrast, the specific activities of the other brush border and intracellular enzymes, including ICFC, were similar in all three segments. This is the first evidence that BBFC, like sucrase, adapts to luminal nutrition. The difference in response of BBFC and ICFC to diet and/or pancreato-biliary secretions provides evidence that these are distinct enzymes which are regulated by different mechanisms.