Translation of mitochondrial cytochrome b RNA in yeast requires the product of the nuclear gene CBS1, a 27.5 kDa soluble mitochondrial protein. In this paper we show that the CBS1 gene is located on chromosome IV immediately adjacent to COX9, the gene coding for cytochrome c oxidase subunit VIIa. CBS1 is transcribed as a very low abundant 900 b RNA. Transcription starts at a single position 101 bp upstream of the CBS1 initiation codon. At positions-39 to-27 of its leader sequence it contains a small open reading frame of 4 codons. By monitoring the β-galactosidase activity of a CBS1/lacZ fusion construct we show that expression of CBS1 is subjected to regulation by oxygen and by glucose: the β-galactosidase activity is elevated threefold in glycerol or galactose grown cells compared to that in glucose grown cells. A further threefold reduction of the activity is observed in anaerobically grown cells. In accordance with this result is the observation that the steady-state level of CBS1 mRNA of anaerobically grown cells is ninefold lower than that of aerobically cultured cells.