A major portion of the intracellular hydrolytic reactions are confined to lysosomes, whose membrane proteins take care of the proper lumenal milieu and the release of the degradation products. To obtain material for structural characterization of the transport proteins, we elaborated a procedure for isolation of lysosomal membranes and separation of their proteins in a two-dimensional electrophoresis. In a first step dense lysosomes were isolated from human placenta using a Percoll gradient. Subsequently, lysosomal membranes were purified by immunoadsorption. The procedure yielded mg-amounts of lysosomal membranes. Proteins associated with lysosomal membranes, acid beta-glucosidase, acetyl-coenzyme A:alpha-glucosaminide N-acetyltransferase, CD63/LIMP I, and h-lamp-2 were enriched approximately 300-fold as compared to the initial homogenate. Separation of the membrane proteins was achieved in a two-dimensional electrophoresis. The procedure is expected to yield material for structural studies on lysosomal membrane proteins with suspected defects in lysosomal storage diseases.