POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-ΔPE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.