GH3 cells which had been exposed for 30 min to [3H]-TRH and had performed their maximum biological response, i.e. increase of prolactin (PRL) release, retained intracellular radioactivity which consisted of chemically unmodified TRH (>93%). Such preloaded GH3 cells were found able to spontaneously release into the culture medium a radioactive material [3H]-RM) which was analyzed. The kinetics of binding of [3H]-RM to intact GH3 cells and competition with unlabeled TRH were indistinguishable from [3H]-TRH. [3H]-RM was able to stimulate PRL release from GH3 cells. In addition thin layer electrophoresis (TLE) revealed that 90% of [3H]-RM migrated like TRH reference. All these features strongly suggest that [3H]-RM is identical to [3H]-TRH.