In a number of dicotyledonous plants, including Arabidopsis, the transcription of organellar genes is performed by three nuclear-encoded RNA polymerases, RPOTm, RPOTmp, and RPOTp. RPOTmp is a protein with a dual targeting, which is presumably involved in the control of gene expression in both mitochondria and chloroplasts. A previous study of the Arabidopsis insertion rpotmp mutant showed that it has retarded growth and development, altered leaf morphology, changed expression of mitochondrial and probably some chloroplast genes, and decreased activities of the mitochondrial respiratory complexes. To date, there is no clear evidence as to which of these disorders are associated with a lack of RPOTmp in each of the two organelles. The aim of this study was to elucidate the role that this RNA polymerase specifically plays in mitochondria and chloroplasts. Two sets of Arabidopsis transgenic lines with complementation of RPOTmp function in either mitochondria or chloroplasts were obtained. It was found that the recovery of RPOTmp RNA polymerase activity in chloroplasts, although restoring the transcription from the RPOTmp-specific PC promoter, did not lead to compensation of the mutant growth defects. In contrast, the rpotmp plants expressing RPOTmp with mitochondrial targeting restored the level of mitochondrial transcripts and exhibit a phenotype resembling that of the wild-type plants. We conclude that despite its localization in two cell compartments, Arabidopsis RPOTmp plays an important role in mitochondria, but not in chloroplasts.