T-cell activation involves multiple signaling pathways. In this report, we conducted in vitro and in vivo immune function analysis of tumor-draining lymph node (TDLN) cells after anti-CD3/anti-CD28 activation versus anti-CD3 activation alone in a murine tumor model. In cytokine release assays, the doubly activated TDLN cells secreted significantly greater amounts of IFN-&ggr; and GM-CSF in response to specific tumor antigen compared with anti-CD3 activated cells. In adoptive immunotherapy, the doubly activated TDLN cells were more effective in mediating regression of 3-day pulmonary metastases compared with anti-CD3 activated cells. Although there was predominant proliferation of CD8+cells after either activation procedure, the mean-fold expansion of CD4+cells was significantly greater after anti-CD3/anti-CD28 activation than anti-CD3 activation alone. Using magnetic bead-enriched T-cell subsets, we found that either CD4+or CD8+doubly activated TDLN cells could independently mediate tumor regression. Furthermore, the doubly activated CD4+cells were more effective than CD8+cells in adoptive immunotherapy on a per-cell basis. The antitumor activity mediated by CD4+or CD8+cells could be significantly enhanced with the exogenous administration of IL-2. CD28 co-stimulation of tumor-primed lymphoid cells promotes the generation of potent tumor reactive effector cells, particularly CD4+T cells, with antitumor activity in adoptive immunotherapy.