The mitochondrial regulatory region (mrr) located between the tRNAphe and tRNApro genes of mitochondrial DNA (mtDNA) is essential for regulation of replication and transcription of the mitochondrial genome. Polyadenylated short RNAs complementary to the L-strand of the mrr in human cells and similar RNAs (polyadenylation status unknown) in rat and mouse cells have been reported. We now report detection of ca. 0.2 kb polyadenylated mrrRNAs in cultured cells of Chinese hamster, African green monkey, mouse, rat, and human. We isolated a cDNA clone to a rat polyadenylated mrrRNA of 158 bp in length excluding the polyadenyl tail, which spans the region from the light strand promoter (LSP) to the origin of heavy strand replication (OriH). This cDNA contains both an open reading frame encoding a 26 amino acid polypeptide and a 12 nucleotide sequence complementary to the 3'-terminus of rat mitochondrial 12S rRNA. A cDNA clone to a human HeLa cell polyadenylated mrrRNA also contains a 12 nucleotide region complementary to the human mitochondrial 12S rRNA. We used a mitochondrial genome-deficient HeLa cell line, ρ° HeLa, and a derived cybrid cell line, HeEB, with a reconstituted mitochondrial genome, to demonstrate that the occurrence of the mrrRNA is dependent on the presence of a mitochondrial genome, and these polyadenylated mrrRNAs are transcribed from the mitochondrial genome. Our results further substantiate the common existence of polyadenylated mrrRNAs among mammals and support previously proposed hypotheses for the multi-functional nature of polyadenylated mrrRNA.