Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.