The effects of microtubule and microfilament inhibitors on ooplasmic segregation and microtubule organization were examined during fertilization, parthenogenetic activation, and early development in the ascidian Molgula occidentalis. At fertilization the egg cortex contracts as the first phase movement and shortly after mitochondria migrate as the myoplasmic crescent develops in the second phase. The microtubule inhibitors colcemid and nocodazole inhibit the second phase, but not the first phase, of ooplasmic segregation. The microfilament inhibitor cytochalasin E has the reciprocal effect of inhibiting the first, but not the second, phase. It appears that sperm may initially bind at any site on the egg surface and that the contractile activities at the first phase and during polar body formation occur independent of the microtubule system. Since the second phase migration occurs as the sperm astral microtubules assemble and since microtubule, but not microfilament, inhibitors arrest this aspect of ooplasmic segregation, microtubules appear necessary for mitochondrial migration. These results demonstrate that the two phases of ascidian ooplasmic segregation are mediated by different systems, the first by microfilaments and the second by microtubules. The microtubule and microfilament systems appear to operate independent of one another and their combined actions result in the completion of ooplasmic segregation. A model is proposed in which the cortical contraction following fertilization is important not only as the motive force for the first phase movement but also as a method to unite the myoplasm with the entering sperm which can initially bind anywhere on the egg surface. The association between myoplasmic components and the growing sperm aster would ensure that the migration and the spatial distribution of myoplasm in the second phase results in the formation of the myoplasmic crescent.