We investigated the mitochondrial behavior, localization and heteroplasmy in reconstituted oocytes derived from germinal vesicle (GV) transfers. The karyoplast containing the GV nucleus and the ooplast (enucleated oocyte) were prepared from GV oocytes derived from B6D2F1 mice. Mitochondria in the karyoplast and ooplast were labeled with MitoRed (Dojindo Laboratories, Kummoto, Japan) and MitoTracker Green (Molecular Probes, Eugene, OR, USA), respectively. After labeling the mitochondria, the karyoplast and ooplast were paired and fused. The mitochondrial behavior in fused (reconstituted) oocytes during in vitro maturation and preimplantation development were observed using confocal laser-scanning microscopy. In reconstituted oocytes that had reached to the M-II stage, mitochondria localized and concentrated in the hemispherical area of oocytes containing the M-II spindle. We showed that the two types of mitochondria derived from the GV donor and the recipient in reconstituted oocytes exhibit similar behavior to the normal oocyte during meiosis, and that the mitochondrial heteroplasmy of these oocytes did not influence their in vitro maturation and preimplantation development.