Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin beta negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin beta is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin beta down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin beta to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin beta 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin beta-regulation of NPC assembly. Excess full-length importin beta and beta 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin beta NPC block, which maps downstream of GTPgammaS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 microM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin beta. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.