The aim of this work is to determine when and how ooplasmic segregation is initiated in the zebrafish egg. To this end, the organization of the ooplasm and vitelloplasm were examined in oocytes and eggs shortly after activation. Ooplasmic segregation, initiated in the stage V oocyte, led to the formation of ooplasmic domains rich in organelles, and ribonucleoproteins. A linear array of closely arranged peripheral yolk globules separated an outer domain of ectoplasm from an inner domain of interconnected endoplasmic lacunae. The structure of this yolk array and the distribution of microinjected labeled tracers suggests that it may provide a barrier limiting ooplasm transit. Loosely arranged yolk globules at the animal hemisphere allow wide connections between the endoplasm and a preblastodisc domain. Activation caused further segregation of ooplasm, reorganization of endoplasmic lacunae, and blastodisc growth. The presence of an endoplasmic cytoskeleton suggests that these changes may be driven by microtubules and microfilaments. Developmental Dynamics 235:656–671, 2006. © 2006 Wiley-Liss, Inc.