In zebrafish (Danio rerio), meroblastic cleavages generate an embryo in which blastomeres cover the animal pole of a large yolk cell. At the 500–1000 cell stage, the marginal blastomeres fuse with the yolk cell forming the yolk syncytial layer. During epiboly the blastoderm and the yolk syncytial layer spread toward the vegetal pole. We have studied developmental changes in organization and function during epiboly of two distinct microtubule arrays located in the cortical cytoplasm of the yolk cell. In the anuclear yolk cytoplasmic layer, an array of microtubules extends along the animal-vegetal axis to the vegetal pole. In the early blastula the yolk cytoplasmic layer microtubules appear to originate from the marginal blastomeres. Once formed, the yolk syncytial layer exhibits its own network of intercrossing mitotic or interphase microtubules. The microtubules of the yolk cytoplasmic layer emanate from the microtubule network of the syncytial layer. At the onset of epiboly, the external yolk syncytial layer narrows, the syncytial nuclei become tightly packed and the network of intercrossing microtubules surrounding them becomes denser. Soon after, there is a vegetal expansion of the blastoderm and of the yolk syncytial layer with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of animal-vegetal microtubules becomes shorter. We investigated the involvement of microtubules in epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated with nocodazole, microtubules were absent and epibolic movements of the yolk syncytial nuclei were blocked. In contrast, the vegetal expansion of the enveloping layer and deep cells was only partially inhibited. The process of endocytosis, proposed to play a major role in epiboly of the yolk syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986) Am. Zool. 26, 193–199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to a delay in all epibolic movements. We propose that the yolk cell microtubules contribute either directly or indirectly to all epibolic movements. However, the epibolic movements of the yolk syncytial layer nuclei and of the blastoderm are not coupled, and only movements of the yolk syncytial nuclei are absolutely dependent on microtubules. We hypothesize that the microtubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the mechanisms by which the two microtubule arrays might function during epiboly are discussed.