When thyroid cells in vitro are stimulated by TSH for 2 h cyclic AMP (cAMP) is synthesized at a high rate during the first 25 min of stimulation, thereafter at a lower rate. The mechanism for this reduction of adenyl cyclase activity was studied in vitro using mouse thyroid tissue. As some of the cAMP formed is released from the cells the sum of cAMP in tissue and incubation medium was studied and considered to reflect the accumulated cAMP synthesis as breakdown synthesis as breakdown by phosphodiesterase was minimized by 1 mM theophylline. The TSH stimulated tissue did not release any adenyl cyclase inhibiting factor into the incubation medium. Cycloheximide, 5 micrograms/ml, enhanced the cAMP response to a single or repeated stimulation by TSH. Its effect was visible after 25 min of incubation and remained at about the same size for 2 h, but the levelling off of cAMP synthesis was not prevented but took place at a higher level. The effect of puromycin, 500 microgram/ml, was similar to that of cycloheximide. Also actinomycin D, 1 microgram/ml, enhanced the cAMP response to TSH. It is concluded that a protein synthesis dependent inhibitor of adenyl cyclase is activated by TSH, but it accounts only for a minor part of the adenyl cyclase inhibition that takes place in the later part of a TSH stimulation of the thyroid. Additional adenyl cyclase inhibiting mechanisms must be considered.