Iodothyronine type II 5'-deiodinase (5'D-II) activities were studied in cultures of rat brown adipocytes. In the presence of serum, the adrenergically stimulated 5'D-II activities were very low. In the absence of serum, adenosine 3',5'-cyclic monophosphate (cAMP) analogues stimulated 5'D-II activity. Thyroxine (T4) inhibited these increases. Norepinephrine slightly increased 5'D-II activity in hypothyroid conditions, but 3,5,3'-triiodothyronine (T3) strongly potentiated the adrenergic stimulation of 5'D-II (20-fold). T3 amplification of the adrenergic stimulation was via beta-adrenergic receptors, specifically mimicked by beta3-agonists, but it was not observed using cAMP analogues. The stimulatory effect of T3 predominated over the inhibitory action of T4, increased with exposure to T3, and required de novo protein synthesis. The half-life of 5'D-II was 30 min, suggesting that stabilization of 5'D-II did not occur. The effect was only observed in differentiated adipocytes. Retinoic acid has similar although smaller effects than T3. In conclusion, the presence of T3 is required and strongly potentiates the noradrenergic stimulation of 5'D-II activity in rat brown adipocytes.