The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98؊͞؊ cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98؊͞؊ cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.T ransport between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE). NPCs have an 8-fold rotational symmetry in the plane parallel to the NE (reviewed in ref. 1). Each NPC contains a membrane-embedded central framework that embraces a central pore. The central framework consists of a ring-like spoke complex that is sandwiched between a cytoplasmic and a nuclear ring. Each cytoplasmic ring supports eight fibrils that extend into the cytoplasm, whereas each nuclear ring carries eight filaments that join distally to form a basket-like assembly (1, 2). The vertebrate NPC has an estimated molecular mass of Ϸ125 MDa and is composed of Ϸ80-100 different proteins called nucleoporins, of which 16-20 have been cloned (3). In contrast, the Saccharomyces cerevisiae NPC is only Ϸ66 MDa (4) and is composed of Ϸ35-50 nucleoporins, of which more than 30 have now been identified (5).Nucleocytoplasmic transport is mediated by soluble transport factors that belong to the karyopherin family of transport receptors, whose members can be subdivided into importins and exportins. Typically, a given transport receptor binds to an import or export signal-containing cargo in one compartment, guides it to and accompanies it through an NPC, releases it in the opposite compartment, and then shuttles back to the first compartment to repeat the cycle (see r...