CD4+ T helper cells may play a critical role in the induction and maintenance of a therapeutic immune response to cancer. To evaluate the efficacy with which a recombinant tumor-associated protein can induce antigen-reactive CD4+ T cells, we stimulated peripheral blood lymphocytes from patients with melanoma in vitro with the purified melanoma antigen gp100 produced in Escherichia coli. In preliminary experiments, we observed that peripheral blood mononuclear cells could process and present known HLA-DRbeta1*0401 and HLA-DRbeta1*0701 restricted epitopes to gp100-reactive CD4+ T cell lines after being loaded exogenously with protein. Therefore, we used autologous protein-loaded peripheral blood mononuclear cells as antigen presenting cells. From four of nine patients who expressed both HLA-DRbeta1*0401 and HLA-DRbeta1*0701, we raised five gp100-reactive CD4+ T cell populations that secreted TH1 type cytokines in response to exogenously loaded protein as well as target cells that endogenously expressed gp100 and MHC class II molecules, including transfectants and melanoma cells. Four of the five cultures specifically recognized the known HLA-DRbeta1*0401 and HLA-DRbeta1*0701 restricted epitopes gp100:44-59 and gp100:170-190, respectively. The fifth culture, and 30 T cell clones derived from it, specifically recognized a new peptide, gp100:420-435, in the context of HLA-DRbeta1*0701. These results suggest that recombinant tumor-associated proteins may be clinically applicable for the generation of CD4+ T helper cells in active vaccination strategies or adoptive cellular immunotherapies.