LON proteases, which are ATP-dependent and exhibit ATPase activity, are found in bacteria, yeast, and humans. In Escherichia coli, LON is known to regulate gene expression by targeting specific regulatory proteins for degradation. The yeast and human LON proteins are encoded in the nucleus but localize to the mitochondrial matrix. In yeast, LON has been shown to be essential for the maintenance of the integrity of the mitochondrial genome. E. coli Lon has long been known to bind DNA, but we have only recently demonstrated that it binds preferentially to a specific TG-rich double-stranded sequence. We now show that human LON recognizes a very similar site in both the light and heavy chain promoters of the mitochondrial genome, in a region which is involved in regulating both DNA replication and transcription. Unlike E. coli Lon, however, human LON specifically binds to the TG-rich element only when it is presented in the context of a single DNA strand. These findings suggest that the human LON protease might regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters.