Alterations in the permeability of the glomerular basement membrane (GBM) towards native ferritin (NF) and iodinated albumin (125I-BSA) following removal of the major glycosaminoglycans (GAGs) of the GBM, heparan sulfate (HS) and hyaluronic acid (HA), were assessed utilizing the techniques of routine electron microscopy and autoradiography, respectively. Kidneys were incubated with heparinase (to degrade the GAGs of the GBM) and subsequently perfused with either NF or 125I-BSA. Control kidneys, which were not treated with heparinase, showed a low permeability to both tracers, with NF being confined to the lamina rara interna and 125I-BSA exhibiting a low level of passage into the urinary spaces (as indicated by a low density of autoradiographic grains over the urinary spaces). After heparinase treatment there was an increase in the permeability of the GBM such that both NF and l25I-BSA passed through the GBM in larger quantities and entered the urinary spaces. Perfusion of cationized ferritin (CF) into control kidneys revealed this probe to bind to the HS-rich anionic sites present within the GBM. Treatment with heparinase resulted in an abolition of the CF binding thereby indicating that the sites are composed mainly of HS and that HS plays a key role in establishing the permeability properties of the GBM. The changes in the pattern of distribution and density of the anionic sites of the GBM following induction of nephrosis was also studied. Animals were rendered nephrotic by subcutaneous injections of an aminonucleoside of puromycin and their kidneys subsequently perfused with either CF or cationized cytochrome c1. No difference in either the pattern of distribution or density of the anionic sites in the GBM of nephrotic kidneys was observed when compared to nonnephrotic controls; thus indicating that the proteinuria associated with aminonucleoside nephrosis might be due to changes in components of the glomerular capillary wall other than the anionic sites.