A UV-cross-linking assay was used to identify RNA-binding proteins in mammalian mitochondria. A number of these proteins were detected ranging in molecular mass from 15 to 120 kDa. All of the mRNA-binding activities were localized to the matrix except for two proteins which are primarily associated with the inner membrane. None of the polypeptides is specific for binding mitochondrial mRNAs since all bound mRNAs from other sources with comparable efficiency. Some preference for binding mRNA over tRNA or homoribopolymers was observed with several of the proteins. A protein with characteristic pentatricopeptide repeat motifs found in many RNA binding proteins was identified associated with the small subunit of the mitochondrial ribosome.