Testicular autoimmune disease (autoimmune orchitis) develops in mice immunized with testis antigen in adjuvant, occurs spontaneously in dogs, mink, and horses, and is a potential cause of human infertility. This is the first study to investigate autoimmune orchitis using monoclonal T cells. Despite the use of crude tissue antigens, 100% of the T cell lines/clones transferred autoimmune disease of the male gonad to normal syngeneic mice, with pathology that affected the testis, the epididymis and/or the vas deferens. Thus orchitogenic peptides are likely of restricted number and/or of dominant immunogenicity. Upon transfer to normal mice, the mildest and earliest pathology elicited by the cloned T cells invariably occurred in a specific region, the testicular straight tubules. Although testis antigen-derived T cell clones responded preferentially to testis antigen, and sperm antigen-derived clones responded more to sperm antigens, each of the 16 clones respond to both antigens. Thus common orchitogenic antigens exist in these germ cell populations though their quantity may differ in distribution. All orchitogenic T cell lines and clones expressed CD4 and the alpha beta T cell receptor; and when activated, they produced interleukin 2, interferon gamma, and tumor necrosis factor (TNF), but not interleukin 4. This cytokine profile characterizes the Th1 CD4+ T cell subset, known to be responsible for the delayed type immunological reaction. Importantly, since disease transfer was significantly and reproducibly attenuated when recipients were injected with neutralizing antibody to TNF, but not neutralizing antibody to interferon gamma, TNF has been defined as a cytokine important in the pathogenesis of this autoimmune disease.