Abstract: We determined the complete sequence of 71 355-bp-long mitochondrial genome from Saccharomyces paradoxus entirely by direct sequencing of purified mitochondrial DNA (mtDNA). This mtDNA possesses the same features as its close relative Saccharomyces cerevisiae - A + T content 85.9%, set of genes coding for the three components of cytochrome oxidase, cytochrome b, three subunits of ATPase, both ribosomal subunits, gene for ribosomal protein, rnpB gene, tRNA package (24) and yeast genetic code. Genes are interrupted by nine group I and group II introns, two of which are in positions unknown in S. cerevisiae, but recognized in Saccharomyces pastorianus. The gene products are related to S. cerevisiae, and the identity of amino acid residues varies from 100% for cox2 to 83% for rps3. The remarkable differences from S. cerevisiae are (1) different gene order (translocation of trnF-trnT1-trnV-cox3-trnfM-rnpb-trnP and transposition of trnW-rns), (2) occurrence of two unusual GI introns, (3) eight active ori elements, and (4) reduced number of GC clusters and divergent intergenic spacers. Despite these facts, the sequenced S. paradoxus mtDNA introduced to S. cerevisiae was able to support the respiratory function to the same extent as the original mtDNAs.