During ooplasmic segregation in ascidians, the myoplasm moves from its original location in the periphery of the unfertilized egg to the vegetal pole of the zygote. The vegetal cap of myoplasm marks the future site of gastrulation and the dorsal side of the embryo. The purpose of this investigation was to determine the mechanism for polarizing the myoplasm during ooplasmic segregation. To test the possibility that the myoplasm moves toward the sperm and that the vegetal pole is the exclusive site of sperm entry, we examined fertilization in egg fragments of the ascidians Styela plicata and Ciona savignyi. Similar frequencies of fertilization were exhibited by various egg fragments, including animal and vegetal fragments or multiple fragments prepared from the same egg. These results indicate that sperm do not enter the egg exclusively at the vegetal pole. Experiments with egg fragments and constricted eggs, combined with chalk marking of the animal pole, demonstrated that after fertilization the myoplasm segregates parallel to the animal-vegetal axis, usually toward the vegetal end of the cell. Activation of primary oocytes with the Ca2+ ionophore A23187 caused the myoplasm to segregate independently of the animal-vegetal axis. This confirms previous experiments in which eggs aligned along a glass fiber coated with A23187 segregated their myoplasm toward the fiber (W.R. Jeffery, 1982, Science 216, 545-547) and suggests that the intrinsic cue for polarization is a release of sequestered Ca2+ at fertilization. Therefore, it appears that ooplasmic segregation and the dorsal-ventral axis are polarized by maternal factors distributed in a concentration gradient along the animal-vegetal axis of the ascidian egg.