The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.