A variety of Saccharomyces strains were examined for the presence of 2micro DNA and, if present, for the pattern of fragments produced by its digestion with site-specific (restriction) endonucleases. Two strains were found that did not contain detectable levels of 2micro DNA, and two strains contained 2micro DNA molecules having only one EcoRI restriction endonuclease recognition site rather than the usual two.-A haploid containing 2micro DNA with one EcoRI restriction site was mated with a haploid containing 2micro DNA with two EcoRI restriction sites and the resulting diploid maintained both types during vegetative growth. Sporulation of the diploid produced four spores, and the clones from these spores contained both types.-A haploid lacking 2micro DNA was mated with a haploid containing 2micro DNA and the resulting diploid contained 2micro DNA. The four clones derived from the haploid spores after sporulation of this diploid all contained 2micro DNA. A rho(-) strain without 2micro DNA was mated to a rho(+) strain with 2micro DNA, and heteroplasmons were selected that had received the nucleus from the strain without 2micro DNA and the mitochondria from the strain with 2micro DNA. Twelve of twenty-four such clones contained 2micro DNA.-I conclude that: (1) the different types of 2micro DNA identified in these strains do not restrict one another, (2) the different types are inherited extrachromosomally, (3) lack of 2micro DNA in two strains is not due to the absence of genes needed for maintenance and (4) the approximately 100 copies of 2micro DNA contained within a single cell are probably clustered within one or a few cytoplasmic organelles.