The mouse mitochondrial DNA genome has been cloned in Escherichia coli by linking it to the pSC101 plasmid replicon at cohesive-ended cleavage sites generated by Eco Rl restriction endonuclease. The four possible configurations of chimeric molecules that contain the nucleotide sequences of mitochondrial DNA in their native relationship were distinguished by Hind III restriction endonuclease digestion and electron microscopic heteroduplex analysis. Chimeric molecules utilize the pSC101 replication origin and do not maintain the "D-loop" region or the low frequency of ribonucleotides found in native mitochondrial DNA. Hybridization of the RNA synthesized in E. coli minicells carrying the four types of chimeras indicates that transcription occurs predominately on the light strand of the mitochondrial DNA in all cases. This result implies that initiation of RNA synthesis occurs within the mitochondrial DNA segment. Although specific polypeptide synthetis is directed by the mitochondrial DNA segment of each of the chimeras in E. coli minicells, the molecular weight distribution of the polypeptides synthesized consists primarily of low molecular weight species and thus differs from that observed in mitochondria in mouse L cells.