Generation of tumor-reactive T cells in large numbers ex vivo is a requisite step in the adoptive immunotherapy of patients. We examined the immune responses of T cells derived from tumor vaccine-primed lymph nodes activated with anti-CD3 alone and with an anti-CD3/anti-CD28 combination. Nylon wool-purified CD3⁺ cells were isolated from vaccine-primed lymph nodes obtained from melanoma, renal cell, and head and neck cancer patients. In the absence of antigen-presenting cells, activation with anti-CD3/anti-CD28 greatly enhanced subsequent T-cell expansion in interleukin 2 (>100-fold), compared to anti-CD3 alone. CD4⁺ T cells were preferentially stimulated. In four of eight patients, we found evidence of CD4⁺ cellular responses to autologous tumors by cytokine release assays. Positively selected CD4⁺ cells activated with anti-CD3/anti-CD28 released greater amounts of cytokine (IFN-γ and granulocyte macrophage colony-stimulating factor) in response to autologous tumors compared to cells activated by anti-CD3 alone. The CD4⁺ reactivity was MHC class II restricted and appeared to be associated with the expression of class II molecules on the vaccinating tumor cells. The CD4⁺ T-cell responses to class II-restricted tumor-associated antigens in patients with renal cell cancers represent unique findings.