AbstractWe report that, during epiboly in zebrafish, three F‐actin–based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring‐like F‐actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo‐BAPTA results in the disruption of all three of these actin‐based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca2+, as well as the functions of other previously described F‐actin–based structures observed throughout epiboly, are discussed. Developmental Dynamics 231:313–323, 2004.© 2004 Wiley‐Liss, Inc.