We have found that Mt element-binding proteins (MtEBPs) with the same binding capability are present in the nuclei and mitochondria of human, bovine, and rat cells. MtEBPs purified from the human nuclei and mitochondria with Mt element-specific DNA affinity chromatography contained 4 different polypeptides, respectively. A UV-induced DNA cross-linking study showed that 47- or 55-kDa of the nuclear MtEBP recognizes Mt in the 5'-flanking region of the human cytochrome c1 gene and that both 140- and 180-kDa polypeptides of the mitochondrial (mt) MtEBPs bind Mts in the human mt promoter region. Mt MtEBPs recognized a Mt4 element-like sequence within the mt transcription factor A binding site for the heavy-strand promoter. These results suggest a MtEBP-mediated coordination mechanism between nuclear and mt genetic systems.