Mutations of individual cysteine residues at codon 301, 390, 398 and 408 of the thyrotropin receptor (TSHr) to serine resulted in cell surface expression of only C301S and C390S mutants. C390S mutation was a silencing mutation with decreased basal constitutive activity. Although the C301S and C390S mutants did not show any significant TSH binding, they generated cyclic AMP upon TSH stimulation. These mutants were also able to interact with stimulating and blocking anti-TSHr antibodies. In fact, C390S receptor is a more sensitive tool for blocking antibody detection than wild type receptor. Introduction of C390S to activating mutations in the ectodomain (S281N), exloop (I486F) and transmembrane (D633H) segments could not mute/nullify receptor activation. These data indicate that the C390S ectodomain behaves as a more effective inverse agonist on the noisy transmembrane segment and suggest that the basal and activated states of the receptor operate through two independent pathways.