Dietary folates exist as pteroylpolyglutamates (PteGlun) that undergo hydrolysis to pteroylmonoglutamate (PteGlu) forms during the process of intestinal absorption. Using the technique of jejunal perfusion of separately labeled folates, our laboratory has demonstrated that hydrolysis of PteGlun occurs on the surface of the jejunum and is a prerequisite for folate absorption. An intestinal brush border pteroylpolyglutamate hydrolase (BB-PPH) has been identified in human and pig jejunum with characteristics that are distinct from those of an intracellular hydrolase (IC-PPH). Functional parallels of BB-PPH with in vivo hydrolysis of PteGlun in human and pig intestine and the clinical responsiveness of BB-PPH to different disease states indicate that this enzyme plays the major physiological role in folate absorption. Folate malabsorption is found in diseases which affect the jejunal mucosa and in response to various drugs. In most of these clinical conditions, folate malabsorption results from suppression of both of the processes of hydrolysis of PteGlun and jejunal uptake of PteGlu. Ongoing studies in miniature pigs are aimed at definition of the sequence of development of folate malabsorption in chronic alcoholism.