Pretreatment of cultured human thyroid cells with trypsin decreased the cAMP response to bovine TSH (bTSH) (by 50-60%). In striking contrast, in trypsin treated cells the cAMP stimulation by both human TSH (hTSH) and thyroid-stimulating antibodies (TSab) was unimpaired, indicating a similar behavior for these two stimulators. The effect of trypsin on inhibiting cAMP stimulation by bTSH was: 1) dose dependent; 2) present at a trypsin concentration as low as 3.3 mg/L; 3) fully reversible within 24 h after removal of the enzyme. In accordance with the altered biological activity in human thyroid cells exposed to trypsin the binding of labeled bTSH was reduced (about 40%). On the contrary, in the same cells, the binding of labeled human TSH was enhanced (about 3-fold). The cAMP response to cholera toxin and forskolin was unaffected in trypsin treated cells, indicating that the tryptic treatment did not alter any other component of the adenylate cyclase complex. The medium obtained from trypsin-treated human thyroid cells was able to neutralize the biological activity of bTSH but not that of hTSH or TSab. Our study demonstrates that in human thyroid cells: 1) trypsin impaires bovine, but not human TSH or TSab biological activity; 2) bovine and human TSH may bind to different components of the TSH receptor.