1 Incorporation of the four labeled ribonucleoside triphosphate into rat liver mitochondrial RNA has been measured. 2 Using 32P-labeled nucleotide as precursor, nearest-neighbour analysis of isolated RNA indicates that the labelling does not result from the formation of homopolymer since all four ribonucleoside monophosphates are found to be labeled. 3 Synthesis of RNA in rat liver mitochondria is inhibited by actinomycin D and by low concentrations of acriflavin indicating that mitochondrial DNA is involved in this process. 4 RNA synthesized in vitro by rat liver mitochondria, during a pulse of 10 min in the presenceof a labeled precursor, has been characterized by sucrose-density centrifugation analysis. RNA synthesized by intact organelle appears to be similar to RNA made by mitochondria with altered permeability of membrane. 5 [3H]AMP-labeled RNA after alkaline hydrolysis showed a higher percentage of radio-activity in the nucleoside fraction than RNA labeled with the other ribonucleoside triphosphates. This suggests that in rat liver mitochondria there may be present also an enzyme responsible for terminal addition of adenylate to transfer RNA molecules.