Mammalian mitochondrial D-loop L-strand RNA (DL-RNA) is thought functionally not important because no obvious function has been found so far. In this study, we detected a novel D-loop L-strand RNA (DL-RNA) in mouse mitochondrion by RT-PCR. The L-strand RNA spans the whole D-loop region of mouse mtDNA, and is resistant to RNase A and RNase T1 but not RNase H digestion. After binding of the L-strand RNA to D-loop, the DL-RNA complex can protect the D-loop from digestion by restriction endonuclease HaeⅢ. These results indicate that a novel RNA-DNA triplex hybrid (R-loop) can be formed in mouse mtDNA D-loop region, and that the DL-RNA structure is capable of protecting the D-loop from certain microbial restriction enzyme digestion. And the similar R-loop structure can not be found in Cyt.b gene in control experiment which confirmed this R-loop is not the fleeting structure in RNA transcription. Considering the D-loop represents the control region of mtDNA, the novel triplex DNA-RNA complex may play an important role in mtDNA replication and transcription.