Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.