Chronic ethanol consumption adversely affects the respiratory activity of rat liver mitochondria. It causes increased cellular production of oxygen radical species and selectively decreases mitochondrial glutathione (GSH) levels. Here we show, using Southern hybridization techniques on total rat genomic DNA, that long-term (11-13 months) ethanol feeding, using the Lieber-DeCarli diet, results in a 36% (P <.05; n = 4) decrease in hepatic mitochondrial DNA (mtDNA) levels when compared with paired controls. UV quantitation of mtDNA isolated from hepatic mitochondria showed that chronic ethanol intake (11-13 months) causes a 44% (P <.01; n = 6) decrease in the amount of mtDNA per milligram of mitochondrial protein. No significant decline in mtDNA levels was seen in ethanol-fed animals maintained on the diet for 1 to 5 months. Ethanol feeding caused a 42% (P <.01; n = 4) and a 132% (P <.05; n = 3) increase in 8-hydroxydeoxyguanosine (8-OHdG) formation in mtDNA in animals maintained on the diet for 3 to 6 months and 10 to 11 months, respectively. In addition, agarose gel electrophoresis revealed a 49% increase (P <.05; n = 3) in mtDNA single-strand breaks (SSB) in animals fed ethanol for more than 1 year. These findings suggest that chronic ethanol consumption causes enhanced oxidative damage to mtDNA in older animals along with increased strand breakage, and that this results in its selective removal/degradation by mtDNA repair enzymes.