The accumulation of cyclic AMP (cAMP) in mouse thyroid tissue in response to TSH in the presence of 1 mM theophylline was accompanied by a release of the nucleotide from the tissue into the incubation medium. This cAMP release was almost rectilinearly related to the time of exposure to TSH, and rectilinearly related to the log concentration of TSH in the range 0.1-5 mU/ml. The cAMP release proved to be independent of the pre-incubation time up to 4 h, and took place also in the absence of methylxanthines when the cAMP level was low. The total cAMP accumulation in response to TSH was augmented by different inhibitors of protein synthesis but the fraction of the nucleotide that was retained intracellularly was increased only by puromycin. Dipyridamole had an effect similar to that of puromycin. Depolarization or treatment with ouabain did not change the distribution of cAMP between tissue and medium. It is concluded that the release of cAMP from thyroid tissue stimulated by TSH may take place under physiological conditions, that it seems to be regulated by the actual concentration of TSH, and that it may be of significance for the regulation of the intracellular cAMP level.