Objective: To study the molecular interactions between the thyroid-stimulating hormone (TSH) receptor (TSHR) and a human thyroid-stimulating monoclonal autoantibody (M22). Design: Amino acid mutations were introduced in the variable region gene sequences of M22 and the wild-type (WT) or mutated M22 Fab expressed in Escherichia coli. The ability of WT or mutated M22 Fab to inhibit binding of 125I-TSH or 125I-M22 to the TSHR and to stimulate cyclic adenosine monophosphate (AMP) production in Chinese hamster ovary cells expressing WT TSHRs was studied. Mutated TSHRs were also used in these studies in combination with WT or mutated M22 Fab to further identify interacting residues in the TSHR-M22 complex. Main outcome: Out of 11 amino acid changes in the heavy chain (HC) of M22, 7 had an effect on M22 Fab biological activity, while in the case of 1 mutation the Fab was not expressed. In particular, stimulating activity of M22 Fab mutated at HC residues, D52, D54, and Y56 was markedly reduced. Mutation of M22 light chain (LC) D52 also reduced M22 Fab stimulating activity, while mutations at two further residues (LC D51 and LC D93) showed no effect. Reverse charge mutations at M22 HC D52 and TSHR R80 provided experimental evidence that these two residues interacted strongly with each other. Conclusion: Mutation of both the TSHR and M22 Fab has allowed identification of some residues critical for the receptor-autoantibody interaction. This approach should lead to detailed mapping of the amino acids important for M22 biological activity. © Mary Ann Liebert, Inc.