This study presents a permeabilized cell system for studying nuclear protein import in mammalian cells. The system uses digitonin to permeabilize the plasma membrane, allowing macromolecules to access the nuclear surface while preserving the nuclear envelope. Transport requires the addition of exogenous cytosol, indicating that soluble cytoplasmic factors are essential for nuclear import. A protein containing a nuclear location sequence (NLS) is rapidly accumulated in nuclei, reaching 30-fold higher concentrations than in the surrounding medium within 30 minutes. Nuclear import is specific for functional NLSs and is ATP and temperature dependent. It is inhibited by wheat germ agglutinin (WGA), which binds to nuclear pore complexes. The system also shows that soluble cytoplasmic factors are involved in transport. The study demonstrates that the permeabilized cell system faithfully mimics nuclear protein import in intact cells. It is simple, efficient, and works with various vertebrate cell sources. The system allows for the biochemical analysis of nuclear transport mechanisms. The results show that the nuclear envelope remains intact during transport, and the basic structure of the cell is preserved. The system is useful for studying the biochemical pathway of nuclear transport and may also be used to analyze RNA export from the nucleus. The study also identifies that soluble cytoplasmic factors are required for nuclear import, and that these factors are different between cytosol and permeabilized cells. The system is not dependent on repairing isolated nuclei and can utilize homologous components. The study provides insights into the mechanisms of nuclear transport and the role of soluble cytoplasmic factors in this process.This study presents a permeabilized cell system for studying nuclear protein import in mammalian cells. The system uses digitonin to permeabilize the plasma membrane, allowing macromolecules to access the nuclear surface while preserving the nuclear envelope. Transport requires the addition of exogenous cytosol, indicating that soluble cytoplasmic factors are essential for nuclear import. A protein containing a nuclear location sequence (NLS) is rapidly accumulated in nuclei, reaching 30-fold higher concentrations than in the surrounding medium within 30 minutes. Nuclear import is specific for functional NLSs and is ATP and temperature dependent. It is inhibited by wheat germ agglutinin (WGA), which binds to nuclear pore complexes. The system also shows that soluble cytoplasmic factors are involved in transport. The study demonstrates that the permeabilized cell system faithfully mimics nuclear protein import in intact cells. It is simple, efficient, and works with various vertebrate cell sources. The system allows for the biochemical analysis of nuclear transport mechanisms. The results show that the nuclear envelope remains intact during transport, and the basic structure of the cell is preserved. The system is useful for studying the biochemical pathway of nuclear transport and may also be used to analyze RNA export from the nucleus. The study also identifies that soluble cytoplasmic factors are required for nuclear import, and that these factors are different between cytosol and permeabilized cells. The system is not dependent on repairing isolated nuclei and can utilize homologous components. The study provides insights into the mechanisms of nuclear transport and the role of soluble cytoplasmic factors in this process.