A technique for the rapid isolation of mitochondrial DNA (mtDNA) from animal tissues is described that eliminates the time-consuming separation of nuclear and mtDNAs using cesium chloride gradient ultracentrifugation. The procedure utilizes digestion of the nuclear DNA with DNase, after which lysis of mitochondria and subsequent extraction of proteins results in relatively pure mtDNA. Up to 5 micrograms of mtDNA per gram of liver tissue resulted, a suitable yield for five digests with restriction enzymes and staining with ethidium bromide.