The thyrotrophin (TSH)-dependent and time-related release of cyclic AMP has been characterised in primary monolayer cultures of normal human thyroid cells. Accumulation of cyclic AMP within the incubation medium was detectable within 1 h of exposure of cultures to 5 mU TSH/ml, and increased throughout of subsequent 15 h incubation period, final levels attained being consistently in excess of the corresponding intracellular cyclic AMP levels. Accumulation of cyclic AMP in the incubation medium was dependent on TSH dose, for both short (1 h) and prolonged (16 h) incubations. Moreover, after incubation for 16 h, cyclic AMP levels in the incubation medium were significantly (P less than 0.01) in excess of intracellular levels for each dose of TSH tested above 0.2 mU/ml. In the absence of TSH, accumulation of cyclic AMP in the incubation medium remained low, after both 1 h and 16 h incubation periods. A consideration of these observations suggests that a bioassay based upon the cyclic AMP content of incubation medium samples should provide a more precise detection system for thyroid stimulators than those measuring the intracellular cyclic AMP response, and this has been demonstrated for a cell preparation in which the intracellular response to TSH was minimal.