We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.