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Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.