Overexpression of inhibitors of apoptosis (IAP) is one potential mechanism for tumor cells to evade immune surveillance. To determine whether immune-mediated killing of tumor cells can be enhanced by neutralization of IAP proteins, 2 novel eGFP-Smac fusion proteins (pro-Smac) were introduced into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5). Each fusion protein contained Smac and a cleavage site specific for granzyme B (GrB) or caspase 8, thereby targeting the 2 major killing mechanisms of cytotoxic T-lymphocyte (CTL) and NK cells. Expression of a pro-Smac fusion protein by D5 tumor cells greatly enhanced the susceptibility to killing by lymphokine-activated killer (LAK) cells or purified GrB. GrB-mediated killing was increased to a much greater extent when tumor cells expressed the eGFP-Smac fusion protein with a GrB cleavage site compared to a caspase 8 cleavage site. In contrast, perforin-deficient LAK cells, which lack GrB-mediated cytotoxicity but process normal ligands for death receptors, killed D5 tumor cells expressed pro-Smac with caspase 8 cleavage site more efficiently. Enhanced killing by GrB was also accompanied by processing of the fusion protein and increased caspase-3-like activity. These results indicate that killing of tumor cells can be amplified by targeting cell-mediated cytotoxic mechanisms via expression of pro-Smac fusion proteins.