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1. In order to demonstrate that triiodothyronine affects mitochondrial RNA synthesis by acting on the enzyme component of the DNA. RNA polymerase complex, mitochondrial RNA polymerase from thyroidectomized and hormone-treated rats was purified up to a stage in which activity was dependent on the addition of exogenous template. In these conditions and using different DNAs as templates, the enzyme from hormone-treated animals displayed an activity about double that of the activity of thyroidectomized animals. 2. Measurements of stability of mitochondrial RNA synthesized in vitro suggest, however, that the hormone can act also at the template level in mitochondrial transcription: the RNA population synthesized in vitro from hormone-treated rats is indeed much more enriched in unstable, probably messenger, RNA species. 3. The turnover of mitochondrial messenger RNA is higher after hormone treatment. 4. Adenosine cyclic 3':5'-monophosphate (cAMP) and its dibutyryl derivative added in vitro to mitochondria from thyroidectomized animals do not affect the incorporation of labeled precursor into mitochondrial RNA, suggesting that the level of the cyclic nucleotide in mitochondria is probably not involved in the hormone action. 5. It is concluded from these and previous studies that the thyroid hormone affects more than one parameter in the mitochondrial transcription process. The interrelationship between these events at molecular level remains, however, to be clarified.