Human anti-glomerular basement membrane (GBM) disease strongly associates with HLA-DRB1*15:01. The target autoantigen in this disease is the noncollagenous domain of the alpha3 chain of type IV collagen, alpha3(IV)NC1, but critical early T cell epitopes presented by this human MHC class II molecule are unknown. Here, by immunizing HLA-DRB1*15:01 transgenic mice with whole recombinant alpha3(IV)NC1 and with overlapping alpha3(IV)NC1 peptides, we defined a HLA-DRB1*15:01-restricted alpha3(IV)NC1 T cell epitope (alpha3) with four critical residues. This peptide was not immunogenic in HLA-DRB1*01:01 transgenic or C57BL/6 mice. The T cell epitope is naturally processed from alpha3(IV)NC1. CD4 T cell clones, generated from HLA-DRB1*15:01 transgenic mice and specific for alpha3, transferred disease into naive HLA-DRB1*15:01 transgenic mice, evidenced by the development of necrotizing crescentic GN, albuminuria, renal impairment, and accumulation of CD4 T cells and macrophages in glomeruli. Because Fcgamma receptors are implicated in disease susceptibility, we crossed HLA transgenic mice onto an FcgammaRIIb-deficient background. Immunization with either alpha3 or alpha3(IV)NC1 induced GN in HLA-DRB1*15:01 transgenic FcgammaRIIb-deficient mice, but HLA-DRB1*01:01 transgenic FcgammaRIIb-deficient mice were unaffected. Taken together, these results demonstrate that the HLA-DRB1*15:01-restricted T cell epitope alpha3 can induce T cell responses and injury in anti-GBM GN.