The proteoglycans on the bladder luminal surface that have been implicated in producing impermeability of the urothelium were investigated. Large-scale isolation was effected with bovine bladder, and smaller scale identification was achieved with human bladder. Little difference was noted between bovine and human bladders. About 80 to 90% of the total surface glycosaminoglycan was bound as integral membrane proteins released with 4 M. guanidinium chloride. About 55% of the protein-bound glycosaminoglycan (proteoglycan) was heparan sulfate, 29% was chondroitin sulfate, and the remainder was either not identified or was dermatan sulfate. Four main proteoglycan species (molecular weight between 85 and 240 kD) were seen, with two minor components present. The proteoglycans were present at very high densities on bladder surface, being stacked 5 to 60 deep. This density of charge will produce a bound water layer that may explain the bladder impermeability.