Cytoplasts from single spermatocytes of NZB/BinJ mice wereseparated from the nuclei and individually microinjected into B6D2F1 (C57BL/6 × DNBA/2J)hybrid embryos at the pronuclear stage (20 h after hCG injection). Of 363 zygotes injected, 311 (86%)survived and developed. From these experiments, we transferred 222 embryos into 20 pseudopregnantrecipients. Eighteen (90%) became pregnant and 82 pups were born (37% of transfers). Mitochondrial DNA(mt DNA) from the NZB/BinJ strain lacks a RsaI restriction site and can thus be distinguished fromthe host embryo following PCR amplification. We were unable to detect the transferred mtDNA inblastocysts on day 4–5 after injection. Nor could we detect NZB/BinJ mtDNA in placentae, norin tissues from mice born to host mothers following the transfer of blastocysts that developed frominjected zygotes. Rejection of paternal mitochondria by the embryo normally occurs at the 4- to 8-cellstage in mice and is apparently dependent on mutual recognition between the mitochondria and thenuclear genome. We conclude that this mechanism has probably already developed by the time the germcells have become committed to meiosis.