ZO-1, ZO-2, and ZO-3 are closely related scaffolding proteins that link tight junction (TJ) transmembrane proteins such as claudins, junctional adhesion molecules, and occludin to the actin cytoskeleton. Even though the zonula occludens (ZO) proteins are among the first TJ proteins to have been identified and have undergone extensive biochemical analysis, little is known about the physiological roles of individual ZO proteins in different tissues or during vertebrate development. Here, we show that ZO-3 knockout mice lack an obvious phenotype. In contrast, embryos deficient for ZO-2 die shortly after implantation due to an arrest in early gastrulation. ZO-2(-)(/)(-) embryos show decreased proliferation at embryonic day 6.5 (E6.5) and increased apoptosis at E7.5 compared to wild-type embryos. The asymmetric distribution of prominin and E-cadherin to the apical and lateral plasma membrane domains, respectively, is maintained in cells of ZO-2(-)(/)(-) embryos. However, the architecture of the apical junctional complex is altered, and paracellular permeability of a low-molecular-weight tracer is increased in ZO-2(-/-) embryos. Leaky TJs and, given the association of ZO-2 with connexins and several transcription factors, effects on gap junctions and gene expression, respectively, are likely causes for embryonic lethality. Thus, ZO-2 is required for mouse embryonic development, but ZO-3 is dispensable. This is to our knowledge the first report showing that an individual ZO protein plays a nonredundant and critical role in mammalian development.