Polyploid embryo production is an important technique in generating mice directly from embryonic stem (ES) cells. The present study was designed to assess the effect of different calcium concentrations and electric field intensities on the production of tetraploid embryos with higher developmental potential by electrofusion. Two-cell mouse embryos were electrofused in fusion solution containing different concentrations of calcium ion (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mM). The rates of blastomere fusion, and subsequent cleavage and development of tetraploids to the blastocyst stage were highest when two-cell embryos were electrically stimulated in a fusion medium containing 1.0 mM calcium. Therefore, we tested electric field intensities (0.6, 0.8, 1.0, 1.2 and 1.4 kV/cm) for electrofusion of two-cell embryos and subsequent development to the blastocyst stage in 1.0 mM calcium. The highest rates of fusion and blastocyst formation were observed when the electric field strength was 0.8 kV/cm. The present results showed that mouse two-cell embryos stimulated with 0.8 kV/cm in a fusion medium containing 1.0 mM calcium had the highest rates of fusion and development to the blastocyst stage.