The specificity of the anti-glomerular basement membrane (GBM) antibodies in experimental nephritis in sheep (Steblay's nephritis) was studied and compared with the specificity of antibodies in human anti-GBM nephritis (Goodpasture's syndrome). Sheep were injected monthly with isolated human GBM and antibody reactivities with isolated human and sheep GBM proteins were quantified with ELISA. Expectedly, the sheep had high titers of antibodies against several human GBM antigens. These antibodies remained for the most part in the circulation. In contrast, circulating antibody levels against sheep GBM antigens remained low for a long period of time, but a significant and progressive increase coincided with the development of acute nephritis. These antibodies accumulated in the kidneys of the nephritic sheep and could be eluted from diseased kidneys. They represent auto-antibodies immunologically cross-reacting with antigens of both sheep and human GBM. The specificity of auto-antibodies eluted from the kidneys was analyzed by immunoblotting and ELISA. The major populations reacted with one subunit, termed M2, of the globular domain of collagen IV. The same subunit contains the major antigen in Goodpasture's syndrome. It is concluded that the M2 subunit of the globular domain of collagen IV is recognized by IgG antibodies that primarily bind to the glomerular basement membrane in both Steblay's nephritis and Goodpasture's syndrome, indicating that it is a main nephritogen in both diseases.