Oct-4 is the earliest expressed gene known to encode a transcription factor which is developmentally regulated during mammalian embryogenesis. In order to understand the role of Oct-4 in early murine embryogenesis, we carried out an analysis of the temporal and spatial pattern of protein expression. We report the presence of Oct-4 protein in cultured cells and murine embryos as determined by immunohistochemistry using confocal microscopy. Oct-4 protein is present in both embryonic stem cells and embryonal carcinoma cells, but is down-regulated following differentiation of these cells by culture in the absence of leukemia inhibitory factor or in the presence of retinoic acid, respectively. In embryos, the protein is found at low levels in unfertilized eggs and is localized predominantly to the pro-nuclei upon their formation following fertilization. The protein is present in the nuclei in all cleavage stages, but following differentiation of the trophectoderm at the blastocyst stage, Oct-4 protein is only expressed in the inner cell mass. The pattern of protein expression to this stage correlates well with previously reported in situ hybridization results; however, a striking difference was seen in primitive endoderm cells which had begun to differentiate and migrate along the inner surface of the trophectoderm. In direct contrast to RNA localization studies which demonstrate that there are only low levels of Oct-4 transcripts in primitive endoderm cells, protein expression in these early migrating cells is higher than in the inner cell mass.