Mitochondrial DNA has been studied in a number of eukaryotic organisms. Differences in inter- and intraspecies mitochondrial DNA restriction patterns have been shown to be due to differences in nucleotide sequences and have been used to study evolutionary relationships and the mode of inheritance of the mitochondrial genome. A relatively rapid and efficient method for the extraction of mitochondrial DNA from Candida parapsilosis and other Candida species was developed. Zymolyase was used to induce yeast protoplasts and mitochondrial DNA was extracted from DNase I-treated mitochondrial preparations. Digestion with the restriction endonucleases Eco RI, Hind III and Bam HI yielded the most definitive restriction patterns. The results of the restriction endonuclease analysis were in agreement with the current identification of these organisms. Candida parapsilosis, Candida albicans and Candida kefyr showed different restriction patterns. Eight Candida parapsilosis strains were compared and all had identical fragment patterns. The molecular size was approximately 30 kilobase pairs and the GC content was 33.2%. The results of these experiments demonstrate the potential of a simple molecular technique for the differentiation of yeast species.