A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear-encoded 5s RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5s RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5s RNA is actually located inside the mitochondrial inner membrane. The 5s rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.