Goodpasture's disease is a rare form of glomerulonephritis characterized by the production of autoantibodies to the glomerular basement membrane (GBM). In order to understand the development of autoimmunity to the GBM, it is important to examine mechanisms underlying T cell responses to the autoantigen. A MoAb PI, with the same specificity as patients’ autoantibodies, was used to affinity-purify the antigen from collagenase-digested human GBM. This material was enriched in the NCI domain of the α3 chain of type IV collagen (α3(IV)NC1), known to be the principal target of anti-GBM antibodies, but also contained lower quantities of α4(IV)NC1. In proliferation assays, T cells from 11/14 patients with Goodpasture's disease showed significant responses (SI ± 2·0) to affinity-purified human GBM. Peak responses were demonstrated at 7 or 10 days at antigen concentrations of 10–30 μg/ml. As in other autoimmune disorders, the presence of autoantigen-reactive T cells was also demonstrated in 5/10 healthy volunteers. Tissue typing revealed that all patients possessed HLA-DR2 and/or -DR4 alleles, while normal individuals whose T cells responded possessed DR2 and/or DR7 alleles. The specificity of the T cell response in Goodpasture's disease was further investigated using monomeric components of human GBM purified by gel filtration and reverse phase high performance liquid chromatography (HPLC). Two antigenic monomer pools were obtained, which were shown by amino-terminal sequence analysis to contain α3(IV)NC1 and α4(IV)NC1, respectively. In all patients tested, significant T cell proliferation was observed in response to one or both of these α(IV)NC1 domains. These results demonstrate that patients with Goodpasture's disease possess T cells reactive with autoantigens known to be recognized by anti-GBM antibodies.