The authors have developed an in vitro system using digitonin-permeabilized vertebrate cells to study the biochemical events involved in the transport of macromolecules across the nuclear envelope. The system allows for the study of nuclear protein import without disrupting the nuclear envelope, which is essential for this process. The authors found that exogenous cytosol is required for nuclear import, indicating that soluble cytoplasmic factors are released during digitonin treatment and are necessary for this process. In this reconstituted system, a protein containing a nuclear location signal rapidly accumulates in the nuclei, reaching a 30-fold concentration compared to the surrounding medium within 30 minutes. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature-dependent. Additionally, the accumulation of the transport substrate within the nuclei is inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. These results suggest that the permeabilized cell system accurately reproduces authentic nuclear protein import. The authors also observed that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factors and components in the insoluble permeabilized cell fraction required for nuclear protein import. This system, being simple, efficient, and applicable to various vertebrate sources, will be powerful for investigating the biochemical pathway of nuclear transport.The authors have developed an in vitro system using digitonin-permeabilized vertebrate cells to study the biochemical events involved in the transport of macromolecules across the nuclear envelope. The system allows for the study of nuclear protein import without disrupting the nuclear envelope, which is essential for this process. The authors found that exogenous cytosol is required for nuclear import, indicating that soluble cytoplasmic factors are released during digitonin treatment and are necessary for this process. In this reconstituted system, a protein containing a nuclear location signal rapidly accumulates in the nuclei, reaching a 30-fold concentration compared to the surrounding medium within 30 minutes. Nuclear import is specific for a functional nuclear location sequence, requires ATP and cytosol, and is temperature-dependent. Additionally, the accumulation of the transport substrate within the nuclei is inhibited by wheat germ agglutinin, which binds to nuclear pore complexes and inhibits transport in vivo. These results suggest that the permeabilized cell system accurately reproduces authentic nuclear protein import. The authors also observed that the sulfhydryl alkylating reagent N-ethylmaleimide inactivates both cytosolic factors and components in the insoluble permeabilized cell fraction required for nuclear protein import. This system, being simple, efficient, and applicable to various vertebrate sources, will be powerful for investigating the biochemical pathway of nuclear transport.