Nuclear import is a critical process for the cell: molecules are selectively permitted into the nuclear interior where the sheltered genome resides. The process of nuclear import can be biochemically studied in vitro using nuclei reconstituted from Xenopus egg extract components and Xenopus sperm chromatin. This in vitro system allows for the visualization of nuclear import by monitoring the accumulation of fluorescent nuclear import substrates in the reconstituted nuclei. A powerful aspect of the system is that “biochemically mutant” nuclei can be readily generated, either by immunodepletion of proteins from or addition of proteins to the reaction. This ability allows ascertainment of the role of specific proteins in nuclear import.