Isolated rat liver mitochondria failed to exhibit in vitro incorporation of [14C]-amino acids into actin-like protein. The use of a pulse-labelling technique demonstrated the appearance of [14C]-actin-like protein in the mitochondria of control, cycloheximide-free rats. The actin-like protein was identified by the method of affinity binding on DNAse1-sepharose and by electrophoresis on polyacrylamide gel with sodium dodecyl sulphate. It was shown that mitochondrial actin-like protein is not included among the nine polypeptides synthesized in mitochondria during cycloheximide-induced blockade of cytoplasmic protein synthesis. It was shown that actin-like protein was not desorbed from mitochondria by repeated washing with isotonic sucrose-mannitol medium. The results obtained indicate that the actin-like protein is biosynthesised in the cytoplasmic compartment.