Administration of soluble human interleukin-12 (hIL-12) has been shown to induce a potent anti-tumor response. However, the use of soluble hIL-12 is hindered by its cytotoxicity when systemically administered and the difficulty of transferring multiple genes into primary tumor cells. In this study, we developed a membrane-anchored hIL-12 and expressed it on tumor membrane vesicles to deliver and confine IL-12 to the vaccination site. We constructed a glycolipid-anchored hIL-12 (GPI-hIL-12) by fusing the coding region of p35 and p40 subunits of hIL-12 with the GPI-signal sequence of CD59 at the C-terminal ends. The two subunits were processed correctly and expressed as a GPI-anchored disulfide-linked heterodimeric protein on the cell surface. GPI-hIL-12 cells induced proliferation of activated T cells and augmentation of allogeneic T cell generation in an MLR assay. Purified GPI-hIL-12 was efficiently intercalated onto isolated tumor cell membrane vesicles prepared from various human tumor cell lines. Further, the incorporation of GPI-hIL-12 onto tumor membrane vesicles induced proliferation of T cells and the release of IFN-gamma by activated T cells. Notably, GPI-hIL-12 enhanced the proliferative response initiated by CD80, a principal costimulatory molecule for T cell activation. These studies suggest that tumor membrane vesicles modified with GPI-anchored cytokines can be used to create potent immunogenic tumor vaccines for use in human immunotherapy. Since protein transfer can be used to modify tumor membrane vesicles obtained from surgical specimens, this approach offers a useful alternative to gene therapy as a means of developing cancer vaccines.