While much emphasis has been placed on the role of MHC class I‐restricted CDS⁺ T cells in the recognition of tumor‐specific antigens (Ag), evidence has accumulated that CD4⁺ T cells also play a critical role in the anti‐tumor immune response. However, little information exists on the nature of MHC class II‐restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein‐Barr virus(EBV) transformed B cells to present melanoma‐associated Ag to melanoma‐specific CD4⁺ cells. CD4⁺ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF‐α and GM‐CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4⁺ cells also recognized MHC class II‐compatible EBV‐B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV‐B cells pulsed with autologous non‐transformed cells or a variety of allogeneic tumors or normal cells. B cells pre‐fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody‐blocking studies defined HLA‐DR as the dominant if not exclusive restriction locus in this T‐B interaction, and HLA‐DR genocyping revealed DRBI 0404 to be the probable restriction element. In a second patient, a CD4⁺ T‐cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV‐B; no cross‐reactivity was observed with the other tumor system investigated, nor with autologous CD4⁺ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV‐transformed B cells to MHC class II‐restricted tumor‐specific CD4⁺ T cells. They also provide a model system for direct identification of these tumor‐derived antigens. © 1994 Wiley‐Liss, Inc.