Restriction fragments produced by a complete Sau3A cleavage of Saccharomyces cerevisiae grande mitochondrial DNA were ligated into the yeast-Escherichia coli shuttle vector YIp5 to establish a clone library representing the mitochondrial genome. 30 hybrid plasmids with an average insert size of 1200 bp were chosen at random and tested for the presence of an autonomously replicating sequence (ars). Over two-thirds of these plasmids transformed yeast at high frequency, indicating the mitochondrial genome contains a large number of ars elements. Our calculations suggest there may be over 40 ars elements contained within the mitochondrial DNA with an average spacing of less than 1700 bp. Mapping experiments indicate that ars elements can be found at many locations on the mitochondrial genome, and in the initial example we have tested, the locations of ars elements derived from grande and petite mtDNAs appear to coincide. If we assume that these ars elements represent mitochondrial DNA replication origins used in vivo, these observations would explain in part the fact that petite mtDNAs can be derived from any location on the grande mitochondrial genome.