We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264-272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27-35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53(+)/HLA-A2(+) tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53(+)/HLA-A2(+) tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.