(1) A method was developed to separate normal rat glomerular basement membrane (GBM) into six components by collagenase digestion, urea extraction, and gel filtration.(2) Treatment of rat GBM with collagenase did not alter nephrotoxic antigenicity of the membrane. Further proteolytic digestion with pronase resulted in loss of ability to elicit nephrotoxic antibody. Rat GBM prepared by mild alkali treatment still retained nephrotoxic antigenicity.(3) Antibodies against isolated components, except two which were retained on Sephadex G-75, localized to GBM and induced proteinuria in rats. One of the components retained on Sephadex G-75 produced localizing, non-nephrotoxic antibodies, while the other produced no localizing antibodies. Multiple antigens and common antigenic determinants among membrane components were demonstrated by gel double diffusion.(4) A specific nephrotoxigenic antigen from rat GBM was characterized as a macromolecular glycoprotein containing 10.7% carbohydrate in the form of galactose, mannose, sialic acid, glucosamine, and galactosamine. On analytical ultracentrifugation in Tris buffer, it gave two peaks with sedimentation constants of 5.93 and 3.49 S, while in 8 M urea it appeared as a single symmetrical peak with lower sedimentation constant (1.33 S) suggesting the presence of aggregate forms of the glycoprotein.(5) Dog GBM and collagenase-solubilized components elicited antibodies which localized to rat GBM but failed to produce proteinuria. Antibody to tendon collagen also localized to rat GBM but had no nephrotoxic effect on normal rats. Antibody to collagen fragments obtained from collagenase digestion failed to localize on rat GBM but still cross-reacted with tendon collagen in gel diffusion.