The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed.