We have identified a novel actin-related 60-kDa polypeptide in mammalian cells. The relatedness of this polypeptide to actin is indicated by its affinity for DNase I, two monoclonal anti-actin antibodies, and two independent peptide-specific anti-actin antibodies which bind to actin at around amino acid 244. It is not incorporated into cytoskeletal stress fibers, although it is a stable protein. Its expression (60-kDa polypeptide, pI of 5.4 to 5.5) is inhibited by the K+ ionophore, nonactin, which is known to collapse the energy-dependent translocation of cytoplasmically synthesized proteins into mitochondria.