We have produced five monoclonal autoantibodies (mA-Abs) to thyroglobulin (Tg) and more precisely to one epitope located within the < 10-kDa pTg tryptic fragment suspension capable of inducing experimental autoimmune thyroiditis (EAT). They were selected from spleen cells from CBA/J mouse immunized with the syngeneic cytotoxic T cell hybridoma HTC2. HTC2 cells are specific for one Tg epitope located within the EAT inducer pTg tryptic fragments and are able to prevent EAT induction by pTg. The restricted specificity of the humoral response previously observed in vivo was further demonstrated and defined in vitro at the single cell level. Competitive studies for binding to pTg or to the < 10-kDa pTg tryptic fragments demonstrated that HTC2-induced anti-Tg mA-Abs recognized an epitope(s) located in the < 10-kDa pTg tryptic fragment (as did 3B8G9, one conventional anti-Tg mA-Ab we selected). We ruled out the possibility that HTC2-induced anti-Tg A-Abs belong to the group of the natural A-Abs due to the lack of recognition of actin, dsDNA, TNP-ovalbumin, tubulin, their isotypes (IgG1 or Ig2a), and their affinities (in the 10(-7) M order of magnitude). The results strengthen the hypothesis that T and B cells sharing the same specificity can express similar idiotopes on their respective receptors for antigen. They also demonstrate the existence of a regulatory idiotypic network that could explain the protection from EAT after injection of inactivated HTC2 cells or its anti-clonotypic mAb.