The TSH receptor (TSHR) undergoes intramolecular cleavage of the ectodomain yielding a two-subunit structure on the cell surface. Subsequently, the TSHR ectodomains (the alpha- or A-subunits) are shed from the cell surface. In this study we first confirmed TSHR alpha-subunit shedding from tagged-TSHR transfected Chinese hamster ovary cells. We found that TSH exacerbated this phenomenon of TSHR subunit shedding. The 125I-TSH cross-linking technique has been suggested as useful in the assessment of dynamic changes in TSHR processing. In our hands this technique did not detect any enhancement of cleavage by TSH. However, we found that the cross-linking method had an inherent insensitivity for studying receptor dynamics as exhibited by its inability to detect even major degrees of TSHR down-regulation. We, therefore, used a cell-based, double-antibody, flow cytometric immunoassay to quantitate TSHR cleavage in real time. We then found that different lines of Chinese hamster ovary TSHR cells, when treated with TSH, showed a time- and dose-dependent increase in TSHR cleavage in addition to ectodomain shedding. We previously reported that monoclonal TSHR stimulating antibody (MS-1) did not always act like TSH. In particular, MS-1 did not enhance TSHR cleavage. However, when we used the Fab fragment of MS-1, we were able to induce cleavage in a similar time frame to TSH. These results suggested that the intact bivalent antibody immobilized the TSHRs in their multimeric state and inhibited intramolecular cleavage. In support of these observations, fluorescence recovery after photo bleaching measurements demonstrated a greater increase in TSHR mobility with MS-1 Fab fragments than with the intact MS-1 IgG. In conclusion, these data indicated that monomer formation from multimeric TSHRs might be an important requirement for TSHR cleavage and TSHR ectodomain shedding.