The effects of T3and T4on the iodothyronine 5’-deiodinase (5’-D) activity in FRTL-5 rat thyroid cells were investigated. T3and T4stimulated the 5’-D activity in a dosedependent manner. Kinetic studies showed that the stimulation of the 5’-D by T3was associated with an increase in maximum velocity (Vmax) in [11.9 ± 0.2 (mean ± SE) and 25.4 ± 0.9 pmol I-released/mg protein • min, respectively, in control and cultured with 10-9M T3for four days] but without a change in apparent Michaelis-Menten constant (Km) (94.8 ± 5.3 nM and 105.4 ± 12.1 nM, respectively). Furthermore, cycloheximide (5 µM) completely abolished the stimulatory effect of T3on the 5’-D activity. T3and T4also enhanced the 5’-D activity stimulated by TSH in a dose-dependent manner. Kinetic studies showed that the stimulatory effect of T3on the 5’-D stimulated by TSH was again associated with an increase in Vmax(86.0 ± 4.0 and 166.5 ± 1.9 pmol I-released/mg protein-min, respectively, cultured with 0.3 U/liter TSH and cultured with TSH plus 10-9M T3for four days) without a change in apparent Km(114.0 ± 7.4 nM and 111.6 ± 12.5 nM, respectively). Cycloheximide (5 µM) completely abolished the stimulatory effect of TSH plus T3 on the 5’-D activity. There were no significant differences observed between cells cultured with TSH and with TSH plus T3in either the intra- or extracellular cAMP contents. Furthermore, T3enhanced the 5’-D activity stimulated by (Bu)2cAMP. These results strongly suggest that T3or T4was synergistic with TSH in stimulating the 5’-D activity in FRTL-5 cells, and that cAMP production would be an important component of the synergism. © 1990 by The Endocrine Society.