The effects of T3 and T4 on the iodothyronine 5'-deiodinase (5'-D) activity in FRTL-5 rat thyroid cells were investigated. T3 and T4 stimulated the 5'-D activity in a dose-dependent manner. Kinetic studies showed that the stimulation of the 5'-D by T3 was 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(-9) M T3 for 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 microM) completely abolished the stimulatory effect of T3 on the 5'-D activity. T3 and T4 also enhanced the 5'-D activity stimulated by TSH in a dose-dependent manner. Kinetic studies showed that the stimulatory effect of T3 on 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(-9) M T3 for four days) without a change in apparent Km (114.0 +/- 7.4 nM and 111.6 +/- 12.5 nM, respectively). Cycloheximide (5 microM) 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 T3 in either the intra- or extracellular cAMP contents. Furthermore, T3 enhanced the 5'-D activity stimulated by (Bu)2 cAMP. These results strongly suggest that T3 or T4 was 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.