Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.