Successful adoptive immunotherapy of cancer requires the identification, isolation, and expansion of tumor-specific immune effector cells. A reliable source of tumor-immune lymphocytes is lymph nodes draining a growing tumor. After in vitro stimulation with anti-CD3 and expansion in IL-2, these cells are capable of mediating the regression of established tumors. In the absence of further Ag stimulation, we recently found that the down-regulation of the homing molecule L-selectin could serve as a surrogate marker for isolation of specific tumor-sensitized T cells. The L-selectin(low) (L-selectin-) T cells proliferated more vigorously than unfractionated or L-selectin(high) cells. In adoptive immunotherapy of established intracranial MCA 205 tumors, L-selectin- cells displayed at least 30-fold greater therapeutic efficacy than unfractionated cells. L-selectin(high) cells did not demonstrate any antitumor effects. Activated L-selectin- cells secreted a number of cytokines, including IFN-gamma, IL-2, IL-4, and IL-10, specifically when stimulated with cognate tumor cells. Further analysis revealed that CD4 T cells alone mediated tumor regression and secreted cytokines. Our results thus demonstrate that the purification of L-selectin- cells led to the generation of CD4 immune effector cells with unusually high therapeutic efficacy against chemically induced tumors. The lack of cytotoxicity and the ability to secrete cytokines suggest that these effector CD4 cells mediate antitumor effects through an indirect mechanism similar to the delayed hypersensitivity reaction.