Metabolic effects upon rat liver mitochondria have been observed by others within 2 min of injection of massive amounts of L-T4. Reported here is the subcellular distribution of 2--5 ng high specific activity [125I]L-T3 2 min after ip injection with and without a loading dose of 30 micrograms unlabeled T3. The loading dose did not change the hormone concentration in the livers of normal or thyroidectomized rats, but it decreased the amount of hormone found in the nuclei in both groups and it increased the mitochondrial hormone only in the normals. The amounts of hormone in the nuclear and mitochondrial fractions were considerably higher in hypothyroid animals than in normals. By chromatography, over 90% of mitochondrial 125I was T3. The 125I content of Triton X-100 extracts of mitochondrial membranes was not affected by stable T3 in vivo or in vitro. It was concluded that specific high affinity, low capacity mitochondrial binding sites could not be found 2 min after hormone injection when specific nuclear binding sites are already evident. Specific binding may not be essential for the rapid effects of thyroid hormones upon rat liver mitochondria.