The effects of specific vitamin deficiencies on the peripheral metabolism of thyroxine (T4) in the rat were assessed. Rats were maintained on a vitamin-free test diet for up to 4 months. Control rats received the diet fully fortified with vitamins. Other groups were fed the diet either unfortified, or completely fortified except for one of the following vitamins: A, riboflavin (B2), pyridoxine (B6), niacin, D, E or K. Deiodinating activity for T4 was assessed both in vivo and in several tissues in vitro. No effects were observed in animals made deficient in vitamins A, Bg, niacin, D or K. T4-deiodinating activity was greatly decreased in homogenates or slices of liver from riboflavin-deficient animals; no change was observed in other tissues. The decrease in hepatic deiodination of T4 also occurred in riboflavin-deficient rats treated with a maintenance dose of T4. Normal activity was restored by feeding the fully fortified diet for 6 days. Deiodinating activity was greatly increased in preparations of liver and muscle, but not in kidney, heart or brain, from vitamin E-deficient rats. The effect of vitamin E deficiency was reversed by administration of the vitamin. In addition, large doses of tocopherol, either administered in vivo or added to homogenates in vitro, greatly decreased hepatic deiodinating activity in both normal and vitamin E-deficient rats. Attempts to demonstrate altered T4 metabolism in vivo in riboflavin or vitamin E-deficient rats have thus far failed. Neither urinary excretion of I131 following injection of I131-labeled T4 nor serum PBI levels were significantly different from those in controls.