Thyrotropin (TSH) receptor monoclonal antibodies (TSHR mAbs) were obtained from cDNA-immunized NMRI mice. Three mAb immunoglobulin Gs (IgGs) (TSmAbs 1-3) that had distinct V_H and V_L region sequences stimulated cyclic adenosine monophosphate (cAMP) production in isolated porcine thyroid cells greater than 10× basal and as little as 20 ng/mL (0.13 nmol/L) of TSmAb 1 IgG caused a 2× basal stimulation. TSmAb 1 and 2 Fab fragments were also effective stimulators and thyroid-stimulating activities of the IgGs and Fabs were confirmed using TSHR transfected Chinese hamster ovary (CHO) cells. The TSmAbs also inhibited ¹²⁵I-labeled TSH binding to TSHR-coated tubes by 50% or more at concentrations of 1 μg/mL or less and gave 15%-20% inhibition at 20-50 ng/mL. ¹²⁵I-labeled TSmAbs bound to TSHR-coated tubes with high affinity (~10¹⁰ L/mol) and this binding was inhibited by TSHR autoantibodies with both TSH agonist and antagonist activities. Inhibition of labeled TSmAb binding by Graves' sera correlated well with inhibition of TSH binding (r = 0.96; n = 18; p < 0.001 for TSmAb 2). The TSmAbs have considerable potential as (1) new probes for TSHR structure-function studies, (2) reagents for new assays for TSHR autoantibodies, and (3) alternatives to recombinant TSH in various in vivo applications.