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The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.