A new RNA polymerase was found in a rat mitochondrial extract. This enzyme showed strong template preference in vitro for the supercoiled recombinant plasmid consisting of pBR322 and the D-loop region of rat mtDNA carrying the origin of heavy-strand replication. The main products synthesized by the D-loop region were two RNAs of different sizes. Both of these products were light-strand products transcribed from the region upstream from the origin of replication. This specific transcription is discussed in relation to initiation of primer RNA synthesis for heavy-strand replication of rat mtDNA.