In vitro fertilization of mouse ova was examined with modified KRB buffer as a culture medium. Only the sperm from cauda epididymis and ductus deference was able to penetrate the zona pellucida of mouse ova. while the sperm from caput epididymis did not. At the final sperm concentration of 1.5×10⁵ sperm/ml, ova were highly penetrated. In the high sperm concentration range, fertilization rate were decreased. It was suggested that this decrease was due to the anti-fertilization factor contained in the sperm suspension. Epididymal sperm required 40-50 min before penetration into the ova but preincubation of the sperm reduced this period. The difference of time before penetration between epididymal sperm and preincubated sperm shows the time need for capacitation (35-40min). Change of pH caused difference in the fertilization rate. Fertilization hardly occurred at pH 7.0, due to inhibition of penetration although the sperm were capacitated at pH 7.0.