We have isolated a cDNA clone encoding chaperonin 10 from rat liver. The cDNA specifies a protein of 102 amino acids which, when transcribed and translated in vitro, yields a single basic product (pI > 9) that co-migrates exactly with the heat shock inducible cpn10 of rat hepatoma cells during 2D gel-electrophoresis. It is concluded that cpn10, unlike the majority of nuclear-encoded proteins of the mitochondrial matrix, is synthesised without a cleavable targeting signal and that, following removal of the initiating methionine, it becomes acetylated prior to mitochondrial import. Incubation of 3H- or 35S-labelled cpn10 with mitochondria confirms these conclusions and shows that cpn10 is imported into mitochondria in an energy-dependent process which is inhibited by the presence of 2,4-dinitrophenol.