Unfertilized eggs of the medaka, Oryzias latipes were inseminated after being mechanically freed from the chorion. In the first step of penetration, as soon as it reached the vitelline surface, the whole spermatozoon was quickly enclosed by ooplasmic protrusions (fertilization cone) of the vitelline surface. In the second step, the egg plasma membrane fused with various regions of the plasma membrane of the enclosed spermatozoon; finally the sperm flagellum was also incorporated into the ooplasm. Initial disappearance of the nuclear envelope of the sperm with vesiculation at the apical region of the head is followed by dispersal of the sperm chromatin. The nuclear envelope is then reformed by fusion of elongated or flattened vesicles along the margin of dispersing nuclear chromatin. The mature male pronucleus has a large nucleolus within a wrinkled envelope. It seems that the fertilization process in this fish involves some features of that occurring in the marine invertebrate and the mammalian eggs.