In mammals, tetraploid embryos produced from normal diploid embryos by artificial fusion do not develop to term. However, tetraploid embryos become functional placentas when they are aggregated with normal diploid embryonic stem (ES) cells. In this case, fetuses are almost completely derived from ES cells whereas placentas are derived from tetraploid embryos. If ES cells could be established from tetraploid embryos, where do they contribute in chimeras? In this study, we investigated the character of tetraploid ES cells.First, we tried to establish tetraploid ES cells from tetraploid blastocysts that were produced by electrical fusion of C57BL/6-EGFP x PWK F1 two-cell stage embryos. Tetraploid blastocysts have less inner cell mass (ICM) than diploid blastocysts and establishment of ES cell lines have been thought to be difficult. However, in our experiment, some ES cell lines were established with the lower efficiency compared with normal diploid ES cells (tetraploid 22% vs diploid 80%). Perhaps, combination of mouse strains and culture condition (serum-free medium) might have led to the success of establishment. DAPI staining clarified established ES cells contained about 80 chromosomes, indicating they were certainly tetraploid ES cells. The diameter of tetraploid ES cells was significantly longer than diploid ES cells (tetraploid 19.9 μm vs diploid 13.9 μm). In the proliferation of cells, doubling time of tetraploid ES cells was not different from that of diploid ES cells (tetraploid 15.9 h vs diploid 15.7 h). Both cell lines formed densely packed colonies and exhibited alkaline phosphatase activity as ES cell maker. Next, we examined the gene expression level of ES cell makers, Oct3/4, Sox2, Nanog, Stella and Fgf5 genes by quantitative real-time RT-PCR. Surprisingly, a relative expression level of Oct3/4 in tetraploid ES cells was about twice that of diploid ES cells though it is reported that twofold increase in expression of Oct3/4 causes differentiation into primitive endoderm and mesoderm. Similarly, a relative expression level of Sox2 in tetraploid ES cells was significantly higher (about 1.7 times) than that of diploid ES cells. In contrast, Nanog, Stella and Fgf5 expressions of tetraploid ES cells were equivalent to or lower than those of diploid ES cells. Embryoid bodies (EBs) were successfully generated from tetraploid ES cells, indicating they have multipotency. Then, we tried to produce chimeras using both ES cells, but no contributions of tetraploid ES cells were observed in both fetuses and placentas at 13.5 and 19.5 days post coitum, whereas control diploid ES cells contributed to several tissues. This is the first report about establishment of ES cells from tetraploid embryos and analysis of their characters. At least the differences of cell size and gene expression pattern between tetraploid and diploid ES cells were observed in this study. Especially about twofold increase of Oct3/4 and Sox2 genes in tetraploid ES cells could be indispensable for corresponding to twice amounts of targets. Thus expression level of these genes could be more strictly regulated than other genes. Oct3/4 and Sox2 genes synergistically activate Oct–Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, and these genes are essential for establishment of induced pluripotent stem (iPS) cells from somatic cells, supporting our idea.