Selected structural and functional alterations of mitochondria induced by bacterial lipopolysaccharide (LPS) were investigated on the basis of the hypothesis that LPS initiates hepatic mitochondrial DNA (mtDNA) damage by oxidative mechanisms. After a single intraperitoneal injection of Escherichia coli LPS, liver mtDNA copy number decreased, as determined by Southern analysis, within 24 hours relative to nuclear 18S rRNA (p < 0.05). LPS induced a novel oxidant-dependent 3.8-kb mtDNA deletion in the region encoding NADH dehydrogenase subunits 1 and 2 and cytochrome c oxidase subunit I, which correlated with mitochondrial glutathione depletion. Expression of mitochondrial mRNA and transcription of mitochondrial RNA were suppressed, whereas mRNA expression increased for selected nuclear-encoded mitochondrial proteins. Resolution of mtDNA damage was mediated by importation of mitochondrial transcription factor A protein, a central regulator of mtDNA copy number, accompanied by binding of mitochondrial protein extract to the mitochondrial transcription factor A DNA-binding site. Hence, mtDNA integrity and transcriptional capacity after LPS administration appeared to be reinstated by mitochondrial biogenesis. These data provide the first link between LPS-mediated hepatic injury and a specific oxidative mtDNA deletion, which inhibits mitochondrial transcription and is restored by activation of mechanisms that lead to biogenesis.