To define the characteristics of isolated glomerular basement membrane (GBM), immunohistochemical and morphometric analyses have been carried out in rat and human tissues. Site-specific arrays of antigens were identified in detergent-isolated GBM in a distribution similar to that observed in intact kidney. In the human, fibronectin, procollagen IV, and collagen V were observed along the internal aspect of GBM continuous with antigenic sites in the mesangium. Another array of antigens was identified in the GBM but not within the mesangium-Goodpasture's antigen, bovine lens capsule type IV collagen, and amyloid P component. In addition, sites reactive with rabbit antiserum to laminin were present on both sides of the lamina densa as well as within the mesangial region. Actomyosin, a presumed mesangial cell antigen, persisted in the mesangium of isolated GBM. Mesangial matrix was identified in detergent-isolated GBM in an amount equivalent to that present in intact glomeruli. Sonicated GBM contained the same antigens but it was not possible to quantitate the amounts of mesangial material by immunofluorescence or morphometric analysis. The thickness of the lamina densa was greater in sonicated and detergent-treated rat GBM preparations than in native rat kidney. These studies demonstrated that isolated GBM is heterogeneous with respect to its antigenic constituents and in addition contains mesangial matrix, which is morphologically and immunohistochemically distinct from peripheral GBM.