Mammalian parthenogenetic embryos (pE) are not viable due to placental deficiency, presumably resulting from lack of paternally expressed imprinted genes. Pluripotent parthenogenetic embryonic stem (pES) cells derived from pE could advance regenerative medicine by avoiding immuno-rejection and ethical roadblocks. We attempted to explore the epigenetic status of imprinted genes in the generation of pES cells from parthenogenetic blastocysts, and its relationship to pluripotency of pES cells. Pluripotency was evaluated for developmental and differentiation potential in vivo, based on contributions of pES cells to chimeras and development to day 9.5 of pES fetuses complemented by tetraploid embryos (TEC). Consistently, pE and fetuses failed to express paternally expressed imprinted genes, but pES cells expressed those genes in a pattern resembling that of fertilized embryos (fE) and fertilized embryonic stem (fES) cells derived from fE. Like fE and fES cells, but unlike pE or fetuses, pES cells and pES cell–fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1 and U2af1-rs1. Coincidently, global methylation increased in pE but decreased in pES cells, further suggesting dramatic epigenetic reprogramming occurred during isolation and culture of pES cells. Moreover, we identified decreased methylation of Igf2r, Snrpn, and especially U2af1-rs1, in association with increased contributions of pES cells to chimeras. Our data show that in vitro culture changes epigenetic status of imprinted genes during isolation of pES cells from their progenitor embryos and that increased expression of U2af1-rs1 and Snrpn and decreased expression of Igf2r correlate with pluripotency of pES cells.