To document the presence of a low Km rT3 and T4-5'-monodeiodinase (5'MDL; Km in nanomolar concentrations) in the liver and to study its characteristics in comparison with the high Km 5'MD (5'MDH; Km in micromolar concentrations), we incubated rat liver microsomal protein (20 micrograms for rT3 substrate and 200 micrograms for T4 substrate) with 125I-labeled rT3 or T4 and dithiothreitol (DTT; up to 20 mM) for 5 min (for rT3) or 30-120 min (for T4) and determined the amount of 125I liberated during incubation. Pilot studies had shown that the activity of rT3 5'MDH is markedly (greater than or equal to 85%) inhibited in the presence of 2 M NaCl, while the rT3 5'MDL is essentially unaffected, and both low and high Km T4 5'MD are minimally (approximately 20%) inhibited. The representative kinetics of various substrates studied were: Km, 13 nM for rT3 5'MDL, 640 for rT3 5'MDH, 26 for T4 5'MDL, and 3620 for T4 5'MDH; maximum velocity, 0.28 nmol/h.mg protein for rT3 5'MDL, 46 for rT3 5'MDH, 0.002 for T4 5'MDL, and 0.46 for T4 5'MDH. Propylthiouracil and iopanoate inhibited all enzymic activities studied. The relative Ki values (micromolar concentrations) for propylthiouracil were: 7.1 for rT3 5'MDL, 1.5 for rT3 5'MDH, 24 for T4 5'MDL, and 40 for T4 5'MDH; those for iopanoate were 0.4 for rT3 5'MDL, 18 for rT3 5'MDH, 7.0 for T4 5'MDL, and 4.0 for T4 5'MDH. DTT was a potent stimulator of enzyme activities studied; its dose (millimolar concentrations) that caused a 50% maximal stimulation was 0.04 for rT3 5'MDL, 1.0 for rT3 5'MDH, 0.025 for T4 5'MDL, and 0.035 for T4 5'MDH. T4 inhibited rT3 5'-monodeiodination and vice versa. The Ki of T4 was 1.3 microM for rT3 5'MDL and 2.0 for rT3 5'MDH, while that of rT3 was 0.4 for T4 5'MDL and 0.6 for T4 5'MDH. We examined the activity of the hepatic 5'MDL (rT3, 0.5 nM; DTT, 0.06 nM; 2 M NaCl) and 5'MDH (rT3, 0.5 microM; DTT, 20 mM; no NaCl) in groups (six animals per group) of rats that were saline treated (control), thyroidectomized, or hyperthyroid (given T3, 20 micrograms/day for 5 days). The relative values for 5'MDL were (mean +/- SD) 17 +/- 3.0, 4.0 +/- 2.0 (P less than 0.01), and 24 +/- 2.0 (P less than 0.01) pmol/h.mg protein, respectively, whereas those for 5'MDH were 13 +/- 4.0, 3.0 +/- 1.6 (P less than 0.01), and 25 +/- 1.0 (P less than 0.01) nmol/h.mg protein, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)