Glomerular basement membrane was prepared from rat, bovine, and human kidneys using a detergent solubilization procedure, and the polypeptide composition of the membranes was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduced rat glomerular basement membrane resolves into 15 polypeptide components ranging in molecular weight from 25,000 to greater than 300,000. Rat, bovine, and human membrane preparations all yield a similar heterogeneous distribution of polypeptides. The same group of polypeptides was observed for membrane prepared with or without the use of proteolytic inhibitors during the isolation procedure. The detergent solubilization method was compared with the more commonly used sonication method on preparations of rat, humans, and bovine glomeruli. Some differences in the number and relative concentrations of polypeptide components was observed. However, a heterogeneous group of polypeptides was present in both types of preparations. These results contradict the recent claim of Cohen and Surma ((1980) J. Biol. Chem. 255, 1767-1770) that glomerular basement membrane prepared by the detergent method using protease inhibitors does not exhibit polypeptide heterogeneity. Therefore, it is concluded that polypeptide heterogeneity is a characteristic feature of glomerular basement membrane irrespective of the method of isolation, detergent or sonication, with or without the use of proteolytic inhibitors.