The cDNAs for interferon-gamma (IFN-gamma) and allogeneic H-2Kd molecules were transfected into highly metastatic B16F10 melanoma cells (H-2b), and the synergistic effects of the antitumor immune responses by the doubly transfected cells (B16/Kd/IFN-gamma cells) were investigated in C57BL/6 mice (H-2b). The singly transfected B16F10 cells with either IFN-gamma or H-2Kd cDNA (B16/IFN-gamma or B16/Kd cells) were used as controls. The B16/Kd/IFN-gamma cells secreted biologically active IFN-gamma, and strongly expressed both syngeneic and allogeneic MHC class I antigens (H-2Kb and H-2Kd) on the same cell construct. Immunization with the doubly transfected B16/Kd/IFN-gamma cells induced higher anti B16F10 cellular cytotoxic responses than the single transfected B16/IFN-gamma or B16/Kd cells. Lyt-2.2 (CD8)+ T-cells were a major effector cell-type involved in the anti B16F10 responses and their cytotoxic activities were augmented in the immunized mice with the B16/Kd/IFN-gamma cells, as demonstrated by in vitro depletion experiments. The survival period of melanoma-bearing mice treated with the B16/Kd/IFN-gamma cells was significantly longer than that treated with the B16/IFN-gamma or B16/Kd cells. Furthermore, the treatment with the B16/Kd/IFN-gamma cells was capable of greatly inhibiting lung metastasis from small, established B16F10 footpad tumors. These results suggest that the augmented immunotherapeutic potentials can be achieved by the vaccination with IFN-gamma and allogeneic MHC class I genes transfected B16F10 melanoma cells.