Zonula occludens-1 (ZO-1) is one of the earliest identified molecular components of tight junctions. Sequence analysis has placed ZO-1 into the broader membrane-associated guanylate kinase (MAGUK) protein family that contains such diverse members as post-synaptic density 95 (PSD-95), Drosophila discs large tumor suppressor gene product (dlg-A), p55, and TamA. Studies in both vertebrates and invertebrates have established that the MAGUK family is involved in a wide variety of cellular functions. These functions involve the regulation of such cellular processes as: (1) tight junction formation, (2) cell proliferation, (3) cell differentiation, and (4) neuronal synapse transmission. Extending these studies, we report the presence of a ZO-1 homologue in Hydra vulgaris, a member of the Cnidaria, the second oldest phylum of the animal kingdom. Hydra ZO-1 (HZO-1) is encoded by a single messenger RNA (mRNA) of approximately 6.0 kb that contains an open reading frame of 5,085 bp. The 191 kDa predicted protein consists of a characteristic MAGUK domain structure, including three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a guanylate kinase (GUK) domain. Western blot analysis using an antibody generated from a synthetic peptide designed from the HZO-1 sequence confirmed the presence of a Hydra protein of the appropriate mass. While whole mount in situ hybridization determined that HZO-1 mRNA was expressed along the entire longitudinal axis of Hydra, cross-sectional analysis established that HZO-1 mRNA expression was restricted to the ectoderm or outer cell layer of the organism's epithelial bilayer. Consistent with this mRNA expression pattern, immunofluorescence studies localized HZO-1 protein to the apical plasma membrane of ectodermal cells. It is unclear what role HZ0-1 has in the cellular physiology of Hydra; however, immunolocalization studies indicate a conserved plasma membrane-associated function(s), as reported for its counterparts in other invertebrate and vertebrate species. These studies establish that the MAGUK family of proteins with a membrane-associated function arose early during metazoan evolution, even before the divergence of protostomes and deuterostomes.