Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into unified structures. Although the processes that regulate development and maintenance of these apical epithelial surfaces are shared across organs, species, and developmental stages, they remain undefined. Using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we showed that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro. We found two domains of ZO-1, U5 and GuK, are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1– deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. Thus, multiple ZO-1–mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surface.