We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC- associated nuclear envelope herniations and growth inhibition at 37°C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.