Pteroylpolyglutamate hydrolase was isolated from rat intestinal mucosa and purified with the aid of affinity chromatography. The affinity ligand was poly-gamma-glutamic acid (Mr approximately 12,000) derived from Bacillus subtilis. The specific enzymatic activity was increased 2,000-fold over the 100,000 X g supernatant of the mucosal homogenate with a yield of 20%. Sephadex G-200 gel filtration yielded an estimated molecular mass of 80,000 daltons. The isoelectric point was pH 8.2. The pH optimum in acetate buffer containing 1 mM zinc was 4.5. The KM values for pteroylheptaglutamate and pteroyltriglutamate were 0.21 and 0.67 microM, respectively. Polyanionic compounds, poly-gamma-glutamic acid, dextran sulfate, and heparin were noncompetitive inhibitors. Studies of the time course of hydrolysis of synthetic [3H]pteroylheptaglutamate by three separate techniques demonstrated the appearance of [3H]pteroylmonoglutamate, synchronous with substrate cleavage. Intermediate pteroyloligoglutamates were not detected. An endopeptidase-like mode of hydrolysis was further established by identification of a hexaglutamyl peptide as the other reaction product.