Many protocols designed to analyze the intracellular distribution of antigens depend on detergents that permeabilize or semi-permeabilize membranes1 . Whereas Triton X-100 or similar detergents are used to permeabilize all cellular membranes, digitonin can permeabilize preferentially the plasma membrane of mammalian culture cells under conditions that leave the nuclear envelope (NE) intact2 . Digitonin treatment of fixed cells has been important to distinguish between antigens on the nuclear and cytoplasmic side of the NE. Furthermore, incubation of unfixed cells with digitonin is a critical step to analyze nuclear import in vitro3 . Macromolecules move in and out of the nucleus through nuclear pore complexes (NPCs) which are embedded in the NE4-6. For the analysis of nuclear transport in a cell-free system it is therefore mandatory that the NE remains intact during the incubation period. Many in vitro nuclear transport studies were carried out with HeLa cells3; however, for some applications the use of other cell lines is preferable. Here, we compare the permeabilization of NEs in three mammalian cell lines that were treated with digitonin. Our protocol provides a guide to optimize the concentration of digitonin to permeabilize the plasma membrane, but not the NE, of mammalian culture cells.