To investigate whether an increase in the intracellular glutathione disulfide (GSSG) concentration actually regulates T4-5'-deiodination in intact cells, rat hepatocytes in primary culture were exposed to glutathione-oxidizing agents (diamide and tertiary butylhydroperoxide) or vinblastine, and their effects on 5'-deiodination of T4 were studied. Deiodinating activity was determined from the 125I- fraction released from [3',5'-125I]T4 added to the serum-free culture medium. Total glutathione (T-GSH) and GSSG levels were determined enzymatically. Diamide (1 mM) and tertiary butylhydroperoxide (0.5 mM) increased the GSSG fraction to approximately 40% of the T-GSH at 5 min, followed by a rapid decrease in GSSG. Glucose deprivation of the medium caused a greater GSSG level at 5 min, followed by a delayed normalization of the increased GSSG level. T4-5'-deiodinating activity was minimally decreased in hepatocytes exposed to 1 mM diamide in the presence of glucose in the medium, but was significantly inhibited in the absence of glucose. Vinblastine, in contrast, gradually and steadily increased the GSSG fraction, and by 3 h, GSSG exceeded 20% of T-GSH (at 10(-4) M vinblastine). This was accompanied by a significant inhibition of 5'-deiodinating activity. When the enzyme activity was inhibited, the T-GSH level was decreased to 40-80% of the control level, which per se cannot account for the decreased T4-5'-deiodinating activity, as reported previously. These data suggest that the increased GSSG level, but not the T-GSH concentration, modulates T4-5'-deiodination in intact cells, and that glucose stimulates the enzyme activity by maintaining glutathione in the reduced form, probably through supplying NADPH, a cofactor for GSSG reductase.