Mitochondria are involved in a variety of cellular biochemical pathways among which the ATP production by oxidative phosphorylation (OXPHOS) represents the most important function of the organelle. Since mitochondria contain their own genome encoding subunits of the OXPHOS apparatus, mtDNA mutations can cause different mitochondrial diseases. The impact of these mutations can be characterized by the trans-mitochondrial cybrid technique based on mtDNA-depleted cells (ρ(0)) as acceptors of exogenous mitochondria. The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation. Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells. Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.