Mitochondrial DNA (mtDNA) sequence variation in the segment of the D-loop region encompassing the initiation sites for replication and transcription was analyzed in the blood of 277 Italian type 2 diabetes patients and 277 Italian healthy subjects. Compared with the Cambridge Reference Sequence, diabetic patients show a slightly higher propensity to accumulate base changes in this region, with respect to controls, although no significant association can be established between any of the detected changes and the diabetic condition. Subjects, patients and controls, harbouring base changes at the replication origins (positions 57 and 151) and at position 58 were analyzed for mtDNA content. The mtDNA content increased three-four times only in the diabetic patients bearing the m.151C > T transition, whereas in those bearing the m.58T > C change the mtDNA content doubled, independently of the affiliation haplogroup. This result suggests that the m.151C > T transition and, to a lower extent, the m.58T > C might confer to the blood cells of diabetic patients the capability of increasing their mtDNA content, whereas the same transitions have no effect on control subjects.