We introduce a new method that removes portions of the plasma membrane of eukaryotic cells to form semi-intact cells. During preparation, these cells lose their soluble cytoplasmic contents, but retain secretory organelles such as the ER and Golgi complex in an intact form. Transport of protein between the ER and Golgi can be functionally reconstituted in vitro using these semi-intact cells by incubation in the presence of cytosol and ATP. Export of the vesicular stomatitis virus strain tsO45 G protein from the ER in vitro is temperature-sensitive, similar to the result observed in vivo. These cells allow direct access of chemicals and antibodies to the cytoplasmic domain of the cell and may be a widely applicable model system for study of a broad range of problems in cell biology.