Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.