In eukaryotic cells, the enclosure of the genetic information in the nucleus allows the spatial and temporal separation of DNA replication and transcription from cytoplasmic protein synthesis. This compartmentalization not only permits a high level of regulation of these processes but at the same time necessitates a system of selective macromolecular transport between the nucleus and the cytoplasm. Transfer of macromolecules between both compartments is mediated by soluble receptors that interact with components of nuclear pore complexes (NPCs) to move their specific cargos. Transport occurs by way of a great variety of different pathways defined by individual receptors and accessory factors. Often, processes in substrate biogenesis that precede transport concurrently recruit transport factors to substrates, thus making transport responsive to correct and orderly synthesis of substrates. Some current challenges are to understand how transport factor-substrate interactions are controlled and integrated with sequential steps in substrate biogenesis, how large macromolecular complexes are restructured to fit through the NPC channel and to understand how transport factor-NPC interactions lead to actual translocation through the NPC.