Mutations in the COL4A5 gene encoding the alpha 5(IV) chain of type IV collagen have been implicated as the primary defect in X-linked Alport syndrome. Several kinds of mutations have been reported so far, spanning point mutations to complete gene deletions. About 5% of Alport patients, who undergo renal transplantation, develop anti-glomerular basement membrane (GBM) nephritis, causing loss of allograft function. In one such patient, COL4A5 gene deletion was recently identified. In the present study, the GBM constituent, targeted by the anti-GBM alloantibodies from the patient who had complete COL4A5 gene deletion was identified. Its identity was determined on the basis of circulating antibody binding to various GBM constituents, domains of bovine type IV collagen and recombinant NC1 domain of human type IV collagen. These results establish, for the first time, the absence of the alpha 5(IV) chain in Alport GBM and, in the same patient, the production of an alloantibody that is targeted to a different chain of type IV collagen, the alpha 3(IV) chain. These findings provide further support for the hypothesis that: (1) anti-alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in Alport GBM, as reflected by the production of anti-alpha 3(IV) alloantibodies.