A radioimmunoassay for detection of antitubular basement membrane (TBM) antibodies was set up using a human TBM antigen (mol wt, 70,000 daltons), purified after collagenase treatment of the insoluble membrane by preparative polyacrylamide electrophoresis, and labeled with iodine 125. Free labeled antigens were separated from those bound to immunoglobulins by a 20% polyethylene glycol (mol wt, 6,000 daltons) solution. In the presence of normal human or Brown Norway rat sera, less than 10% of the labeled antigens were precipitated. In the presence of sera or of kidney eluates from rats immunized with human TBM, the precipitation of the labeled TBM antigens reached 73%, but in the presence of sera from two patients presenting with an interstitial nephritis and linear deposits along the TBM only, up to 47% of the same antigens were precipitated. In these two cases, the anti-TBM antibodies were mainly directed against the heteropolysaccharide-containing glycopeptides isolated from TBM, that is, against the noncollagenous polypeptides of the TBM antigens. Anti-TBM antibodies were sought in the sera of 52 normal blood donors and of 11 patients presenting with glomerulonephritis and linear deposits of immunoglobulins. The average percentage (+/- 1 SD) of labeled TBM antigens precipitated in the serum of normal blood donors was 7.1 +/- 1.2. Of the patients presenting with glomerulonephritis and linear deposits along the GBM, 9 out of 11 exhibited anti-TBM antibodies by radioimmunoassay; among these 9 patients, 8 also displayed linear deposits of IgG along the TBM. Absorption of anti-TBM and anti-GMB antibodies with particulate TBM or GBM, with both types of glycopeptides isolated from GBM or TBM, indicated that the anti-TBM antibodies were directed against the noncollagenous polypeptides of TBM but that the anti-GBM antibodies mainly reacted with the collagenous polypeptides of TBM and GBM. Finally, it was found that the sera of 2 patients out of 15 presenting with lupus nephritis contained a significant anti-TBM-binding activity, mainly directed against the noncollagenous material of TBM.