We have identified and characterized a cyclic AMP receptor protein in mitochondria of the yeast Saccharomyces cerevisiae. The binding is specific for cyclic nucleotides, particularly for cyclic AMP which is bound with high affinity (Kd of 10(-9) M) at 1 to 5 pmol/mg of mitochondrial protein. The mitochondrial cyclic AMP receptor is synthesized on cytoplasmic ribosomes and has an apparent molecular weight of 45,000 as determined by photoaffinity labeling. It is localized in the inner mitochondrial membrane and faces the intermembrane space. Cross-contamination of mitochondrial inner membranes by plasma membranes or soluble cytoplasmic proteins is excluded.