The effects of thyroid hormone (TH) status on energy metabolism and tissue-specific substrate supply in vivo are incompletely understood. To study the effects of TH status on energy metabolism and tissue-specific fatty acid (FA) fluxes, we used metabolic cages as well as (14)C-labeled FA and (3)H-labeled triglyceride (TG) infusion in rats treated with methimazole and either 0 (hypothyroidism), 1.5 (euthyroidism), or 16.0 (thyrotoxicosis) microg per 100 g/d T(4) for 11 d. Thyrotoxicosis increased total energy expenditure by 38% (P = 0.02), resting energy expenditure by 61% (P = 0.002), and food intake by 18% (P = 0.004). Hypothyroidism tended to decrease total energy expenditure (10%; P = 0.064) and resting energy expenditure (12%; P = 0.025) but did not affect food intake. TH status did not affect spontaneous physical activity. Thyrotoxicosis increased fat oxidation (P = 0.006), whereas hypothyroidism decreased glucose oxidation (P = 0.035). Plasma FA concentration was increased in thyrotoxic but not hypothyroid rats. Thyrotoxicosis increased albumin-bound FA uptake in muscle and white adipose tissue (WAT), whereas hypothyroidism had no effect in any tissue studied, suggesting mass-driven albumin-bound FA uptake. During thyrotoxicosis, TG-derived FA uptake was increased in muscle and heart, unaffected in WAT, and decreased in brown adipose tissue. Conversely, during hypothyroidism TG-derived FA uptake was increased in WAT in association with increased lipoprotein lipase activity but unaffected in oxidative tissues and decreased in liver. In conclusion, TH status determines energy expenditure independently of spontaneous physical activity. The changes in whole-body lipid metabolism are accompanied by tissue-specific changes in TG-derived FA uptake in accordance with hyper- and hypometabolic states induced by thyrotoxicosis and hypothyroidism, respectively.