We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 μM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating ‘uncoupling’ of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the N_i inhibitory pathway of adenylate cyclase by epinephrine (10⁻⁶ M-10⁻⁴ M in the presence of 10⁻⁴ M propranolol) was unaltered in cells previously desensitized to TSH. That is, N_i-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3–4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.