Most eukaryotes maintain multiple copies of mtDNA, ranging from 20-50 in yeast to as many as 10,000 in mammalian cells. The mitochondrial genome encodes essential subunits of the respiratory chain, but the number of mtDNA molecules is apparently in excess of that needed to sustain adequate respiration, as evidenced by the "threshold effect" in mitochondrial diseases. Thus, other selective pressures apparently have contributed to the universal maintenance of multiple mtDNA molecules/cell. Here we analyzed the interplay between the two pathways proposed to regulate mtDNA copy number in Saccharomyces cerevisiae, and the requirement of normal mtDNA copy number for mitochondrial gene expression, respiration, and inheritance. We provide the first direct evidence that upregulation of mtDNA can be achieved by increasing ribonucleotide reductase (RNR) activity via derepression of nuclear RNR gene transcription or elimination of allosteric-feedback regulation. Analysis of rad53 mutant strains also revealed upregulation of mtDNA copy number independent of that resulting from elevated RNR activity. We present evidence that a prolonged cell cycle allows accumulation of mtDNA in these strains. Analysis of multiple strains with increased or decreased mtDNA revealed that mechanisms are in place to prevent significant changes in mitochondrial gene expression and respiration in the face of approximately two-fold alterations in mtDNA copy number. However, depletion of mtDNA in abf2 null strains leads to defective mtDNA inheritance that is partially rescued by replenishing mtDNA via overexpression of RNR1. These results indicate that one role for multiple mtDNA copies is to ensure optimal inheritance of mtDNA during cell division.