The ability to generate a large number of tumor-reactive T lymphocytes is the most critical requirement for adoptive immunotherapy. Our laboratory has previously demonstrated that cells from tumor-draining lymph nodes (LNs) are an excellent source of tumor-reactive T lymphocytes. After activation with anti-CD3, these cells readily proliferate in low concentrations of interleukin 2 and acquire effector functions. The adoptive transfer of these cells is capable of mediating the regression of tumors established in the lung as well as in the brain. Here, we analyzed several adhesion molecules on the tumor-draining LN T cells and separated them based on l-selectin expression. The homing receptor l-selectin mediates adhesion to the luminal surface of specialized high endothelial venules, thus regulating lymphocyte recirculation through peripheral LNs. In response to progressive tumor growth, a small population of draining LN T cells down-regulated l-selectin and increased the expression of CD44 and lymphocyte function-associated antigen 1. In adoptive immunotherapy, purified T cells with low l-selectin (l-selectin^-) expression constituted all the in vivo antitumor reactivity, whereas isolated high l-selectin (l-selectin⁺) cells were ineffective. Furthermore, reverse transcription-PCR analysis revealed that l-selectin^- cells expressed interleukin 2, IFN-γ and tumor necrosis factor α mRNA upon in vitro stimulation with specific tumor cells. These results suggest that highly potent immune T cells can be isolated based on their pattern of adhesion molecule expression. The ability of the immune effector cells to transcribe cytokine genes when stimulated with tumor cells provides a basis for identifying similar cells for adoptive immunotherapy of cancer in humans.