The nuclear gene for a mitochondrial ribosomal protein, termed YMR26, of Saccharomyces cerevisiae strain DC-5 was cloned by hybridization with synthetic oligonucleotide mixtures corresponding to the N-terminal amino acid sequence of this protein. The gene was found to occur in a single copy on either chromosome VII or chromosome XV. The nucleotide sequence of the cloned segment containing this gene showed the presence of an open reading frame capable of encoding a basic protein of 18.5 kDa with 158 amino acid residues. The deduced amino acid sequence showed no significant similarity to any known ribosomal proteins of prokaryotic or eukaryotic origin or to any other proteins in the NBRF protein data bank. When the gene was disrupted by insertion of a 2.9 kb restriction fragment containing LEU2, cells became PET- indicating that the gene is essential for yeast mitochondria. Northern blot analysis indicated that the size of the transcript from the YMR26 gene was approximately 530 nucleotides long. The expression level of the YMR26 gene was monitored upon catabolite repression, in strains with various mitochondrial genetic backgrounds and in strains harboring an increased dosage of the YMR26 gene. In rho+ cells, the transcription of the YMR26 gene was more repressed in a medium with glucose than in the presence of either galactose or nonfermentable carbon sources. However, in rho o cells, its transcription appeared not to be repressed even by high concentrations of glucose. The amount of the YMR26 mRNA was increased 10-fold when cells carried the YMR26 gene on a high-copy number plasmid.