RNA interference is used to specifically and effectively inhibit the expression of cognate genes. In the present study we investigated the inhibitory effect of gene expression in mouse embryos developing in vitro by injecting short interference RNA (siRNA). Fertilized mouse zygotes were obtained from mated females 20–24 h after hCG injection. Chemically synthesized 21-nt siRNA was commercially obtained and injected into mouse zygotes. The zygotes were then cultured in KSOM medium supplemented with 4% BSA at 37°C. Semi-quantitative RT-PCR was used to examine Octamer-binding transcription factor (Oct-4) gene expression in a single mouse embryo developing in vitro following siRNA-injection. In order to determine the expression and distribution of Oct-4 in mouse embryos, the mouse embryos were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.2% triton x-100 for 10 min. Embryos were then incubated with rabbit Oct-4 polyclonal antibody for 1 h and with FITC-labeled goat anti-rabbit antibody. Propidium iodide was used for DNA staining. siRNA injection did not retard the development of mouse zygotes. The number of blastocyst cells and the ICM/TE ratio did not differ in the siRNA injected blastocysts and the non-injected control group. Semi-quantitative RT-PCR revealed that Oct-4 expression was decreased at the 4-cell embryo stage and was significantly high at the morula and blastocyst stages. Injection of siRNA into oocytes inhibited RNA expression of Oct-4 and Nanog, but not of E-cadherin and Heat shock protein 70.1. Immunocytochemical staining showed inhibition of Oct-4 synthesis of the morulae and blastocysts following injection of siRNA. After culture of the embryos in the ES cell-derived conditioned medium, the embryos were stained for alkaline phosphatase (AP), a marker specific to pluripotent cells. AP was not detected in the inner cell mass of blastocysts following siRNA injection. These results suggest that siRNA injection into a mouse zygote specifically inactivates Oct-4 in mouse embryos developing in vitro.