A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.