The aim of the present study was to examine transient expression of transgene injected into nuclei of rat 2-cell stage embryos. We also investigated the relationship between expression in both blastomeres and tail position of penetrated spermatozoa in rat 2-cell stage embryos. Rat 2-cell stage embryos were recovered from superovulated Wistar females mated with same strain mature males at 48 h after hCG injection. DNA fragments, as the transgene containing the EGFP (enhanced green fluorescent protein) gene controlled under the CMV-IE promoter, were microinjected into one nucleus of 2-cell stage embryos. After microinjection, embryos were cultured in KRB at 37.0°C in a 5% CO₂ and 95% humidified air until observation. First, transient EGFP expression in 151 injected embryos was observed using a fluorescence microscope at 6 h intervals until 48 h after injection. At 6 h after microinjection fluorescent embryos were detected, and the proportion of fluorescent embryos increased over time. The rate reached maximum (84%, 52/62) at 24 h after microinjection, and several fluorescent patterns of fluorescent blastomeres in the embryos were observed. There were blastomeres with the same or different fluorescence levels and a single fluorescent blastomere. Second, to assess tail position of the penetrated sperm in the fluorescent embryos, 75 whole mount specimens were observed by inverted phase-contrast microscopy at 24 h after the injection. Also, parthenogenetic 2-cell stage embryos that never contained sperm tail were microinjected with the transgene and observed in the same manner. To obtain parthenogenetic 2-cell embryos, 80 ovulated ova were collected from non-mated females, and incubated with 2 mM 6-DMAP for 4 h. The ova were additionally cultured for 20 h in KRB at 37.0°C in a 5% CO₂ and 95% humidified air. In embryos with both blastomeres fluorescent (94%, 33/35), the sperm tail existed in both blastomeres like a bridge between blastomeres. In contrast, in one embryo with a single fluorescent blastomere (4%, 1/24), the sperm tail existed in both blastomeres, and in other embryos with a single fluorescent blastomere (75%, 18/24), the sperm tail was positioned in the one blastomere. On the other hand, in 63 parthenogenic rat 2-cell embryos in which there was no sperm tail, most embryos (86%, 54/63) had a single fluorescent blastomere at 24 h after microinjection. The results indicated that the sperm tail position in the 2-cell embryos makes the protein migration variable. In conclusion, when the CMV-IE/EGFP gene was microinjected into nuclei of rat 2-cell embryos, at 24 h after the microinjection the EGFP was detected in most embryos; however, fluorescent patterns in blastomeres varied. It seems that EGFP derived from the transgene injected into one blastomere may move into another blastomere in rat 2-cell stage embryos, and that the presence of a sperm tail in both blastomeres may influence EGFP distribution.