Mitochondrial DNA (mtDNA) has been isolated from four strains of Physarum polycephalum and a restriction site map has been determined using nine restriction enzymes. The restriction site maps of the four strains are similar but each strain is distinguished by insertions, deletions and restriction enzyme site polymorphisms. The sum of the restriction fragments gives mitochondrial genome sizes which vary from about 56 kb to 62 kb. In all four strains the composite map of the restriction enzyme sites for the mtDNA is circular. Knowledge of the restriction enzyme map has enabled cloning of mtDNA fragments representing the entire mtDNA of strain M3. The cloned fragments have been used to create a transcription map of the mtDNA.