An efficient method for isolation of acid alpha-glucosidase from human spleen is developed. The method involves chromatography of the enzyme on rho-aminophenyl-alpha-D-glucopyranoside covalently bound to CH-Sepharose 4B, with subsequent gel filtration on Sephadex G-200. The enzyme was homogeneous by the polyacrylamide gel electrophoresis data; it was purified about 1500-fold, as compared with the crude extract (the total yield 12.5%). Besides acid alpha-glucosidase, the preparations of alpha-L-fucosidase, alpha-D-galactosidase and beta-N-acetylglucosaminidase were isolated and purified 200-, 130- and 280-fold, respectively. The nature of interaction between acid alpha-glucosidase and immobilized rho-aminophenyl-alpha-glucopyranoside is discussed.