Previous perfusion studies of the human jejunum suggested that conjugated folate is hydrolyzed on the mucosal surface. The techniques of cell fractionation and DEAE and gel chromatography led to the identification of two separate folate conjugase activities in human jejunal mucosa: one membrane-bound and concentrated in the brush border, the other soluble and intracellular. These enzyme activities exhibit different pH optima, molecular weights, and inhibition characteristics. Folate conjugase in the brush border may accomplish the initial digestion of dietary pteroylpolyglutamates.