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Abstract: In a previous study, we used mouse zygotes as recipients of mtDNA with a large-scale deletion mutation (ΔmtDNA) and generated respiration-deficient mice (mito-mice) carrying ΔmtDNA. In this study, we used mouse ES cells as recipients of ΔmtDNA, and generated mito-mice with ΔmtDNA only when the ES cells carried 17% ΔmtDNA. No chimera mice or their F1 progenies were obtained from ES cells carrying more than 61% ΔmtDNA. These observations suggest that respiratory defects of ES cells inhibit their normal differentiation into chimera mice and mito-mice, and that ES cells are more effective than zygotes for generation of mito-mice carrying mtDNAs without significant pathogenic mutations. [Copyright &y& Elsevier]