The addition of immunostimulatory molecules to tumor cells has been proposed as a potentially useful strategy to induce anti-tumor immunity. In this report we have investigated the application of using isolated tumor membranes modified by transfer of a glycosyl-phosphatidylinositol (GPI)-anchored form of the costimulatory molecule, B7-1 (CD80), as a cell free cancer vaccine for clinical use. Isolated tumor cell membranes were prepared from established tumor cell lines and the optimum conditions necessary for modification and clinical application were determined. GPI-B7-1 transferred optimally onto isolated human tumor membranes at physiological temperature (37 degrees C) in a dose dependent manner. Transfer of GPI-B7-1 to isolated membranes resulted in stable expression and costimulatory function. These modified membranes could be stored for repeated immunizations while retaining expression of GPI-B7-1. Critically, isolated tumor membranes, prepared directly from surgically removed human tumor tissue, could be modified by GPI-B7-1 and costimulate T cells. Finally, membranes isolated from tumor tissue expressed MHC class II, unlike the cell line established in vitro from the same patient. This novel approach to express immunostimulatory molecules on isolated membranes derived from a patient's tumor tissue will make the preparation of autologous therapeutic cancer vaccines available to patients from which tumor cell lines can not be established.