The total nucleotide sequence of the mitochondrial genome of the yeast Kluyveromyces lactis was determined. The DNA is a circular molecule of 40,291 base pairs, with 26.1% GC. It contains a set of protein- and RNA-coding genes equivalent to those of the Saccharomyces cerevisiae mitochondrial genome. The genome size is about one half of that of S. cerevisiae mitochondrial DNA. The difference in size is due essentially to a reduced proportion of intergenic and intronic sequences. The coding sequences occupy about one third of the genome, the rest being composed of AT-rich sequences and numerous short GC-rich clusters that are dispersed mostly in the non-coding regions and a few within coding sequences. The presence of these GC clusters is a characteristic feature common to K. lactis and S. cerevisiae mitochondrial DNA, although their sequence patterns are different. The absence of the NADH dehydrogenase subunit genes distinguishes this yeast and S. cerevisiae from the typically aerobic species. The genetic code appears to be that of the standard fungal mitochondrial genomes, with UGA as a tryptophan codon. There are only 22 transfer RNA genes, those corresponding to CUN and CGN codons being missing. CUN codons are absent in the protein-coding sequences. There are five CGN codons within the open reading frames, but they are located exclusively in the introns, rendering them untranslatable. Introns are found only the genes in KlCOX1 and LrRNA. The transcription promoter motif known in S. cerevisiae and several other yeast species is also present. All genes are transcribed from the same strand, except those on a single 7-kilobase pairs segment (EMBL Accession No. AY654900).