A s judged from both paper and column chromatography, slices or homogenates of liver from rats fasted for 48 h displayed a lesser rate of generation of ¹²⁵I-labeled 3,5,3′-triiodothyronine (T₃) from ¹²⁵I-labeled thyroxine (T₄) added to incubation media than did preparations from normal chow-fed animals. A similar defect in the conversion of T₄ to T₃ in the livers of fasted animals was observed when preparations were incubated with substrate concentrations of T₄ so that T₃ generation could be assessed by radioimmunoassay. The effect of fasting could be prevented, wholly or in part, by administration of glucose in the drinking water to otherwise fasted animals, and the degree of prevention appeared to be proportional to the concentration of glucose employed. Diminished generation of T₃ from T₄ was similarly evident in the livers of animals with streptozotocin-induced diabetes mellitus, and this defect was overcome by the provision of insulin in vivo, but not in vitro. Decreased formation of T₃ from T₄ was also observed in preparations of liver from animals given dexamethasone, amiodarone, and propylthiouracil. In no case could these effects on the net formation of T₃ from T₄ be explained by effects of the experimental conditions on the degradation of the T₃ generated, as judged from the rate of degradation of exogenous ¹²⁵I-T₃ measured in parallel incubates.An analysis of the rate of disappearance of ¹²⁵I-T₄ from reaction mixtures in relation to the rate of appearance of ¹²⁵I-T₃ and ¹²⁵I-iodide was employed to estimate the activity of the 5-monodeiodinating pathway of T₄ metabolism that leads to the formation of 3,3′,5′-triiodothyronine (reverse T₃). Such estimates indicated that reverse T₃ formation was actively proceeding in the preparations studied, was slightly enhanced by fasting, was unaffected by dexamethasone and amiodarone, and was markedly inhibited by propylthiouracil.In view of the similarities between the effect of these experimental manipulations on the generation of T₃ from T₄ by rat liver in vitro to their effects on the production rates and serum concentrations of T₃ in man, it is concluded that the rat liver system provides a suitable model for the study of factors that influence the conversion of T₄ to T₃ in man. In addition, the findings strongly indicate that this process, at least in the liver, is closely linked to the utilization of carbohydrate.