Parthenogenesis is the process by which an oocyte develops into an embryo without being fertilized by a spermatozoon. Although such embryos lack the potential to develop to full term, they can be used to establish parthenogenetic embryonic stem (pES) cells for autologous cell therapy in females without needing to destroy normally competent embryos. Unfortunately, the capacity for further differentiation of these pES cells in vivo is very poor. In this study, we succeeded in improving the potential of pES cells using a nuclear transfer (NT) technique. The original pES cell nuclei were transferred into enucleated oocytes, and the resulting NT embryos were used to establish new NT-pES cell lines. We established 84 such lines successfully (78% from blastocysts, 12% from oocytes). All examined cell lines were positive for several ES cell markers and had a normal extent of karyotypes, except for one original pES cell line and its NT-pES cell derivatives, in which all nuclei were triploid. The DNA methylation status of the differentially methylated domain H19 and differentially methylated region IG did not change after NT. However, the in vivo and in vitro differentiation potentials of NT-pES cells were significantly (two to five times) better than the original pES cells, judged by the production of chimeric mice and by in vitro differentiation into neuronal and mesodermal cell lines. Thus, NT could be used to improve the potential of pES cells and may enhance that of otherwise poor-quality ES cells. It also offers a new tool for studying epigenetics.