Although they are considered as antigen-presenting cells, the role of antigen-unspecific B lymphocytes in antigen presentation and T-lymphocyte stimulation remains controversial. In this paper, we tested the capacity of normal human peripheral activated B cells to stimulate T cells using melanoma antigens or melanoma cell lysates. B lymphocytes activated through CD40 ligation and then pulsed with tumor antigens efficiently processed and presented MHC class II-restricted peptides to specific CD4⁺ T-cell clones. This suggests that CD40-activated B cells have the functional and molecular competence to present MHC class II epitopes when pulsed with exogenous antigens, thereby making them a relevant source of antigen-presenting cells to generate T cells. To test this hypothesis, CD40-activated B cells were pulsed with a lysate prepared from melanoma cells and used to stimulate peripheral autologous T cells. Interestingly, T cells specific to melanoma antigens were generated. Additional analysis of these T-cell clones revealed that they recognized MHC class II-restricted epitopes from tyrosinase, a known melanoma tumor antigen. The efficient antigen presentation by antigen-unspecific activated B cells was correlated with a down-regulation in the expression of HLA-DO, a B cell-specific protein known to interfere with HLA-DM function. Because HLA-DM is important in MHC class II peptide loading, the observed decrease in HLA-DO may partially explain the enhanced antigen presentation after B-cell activation. Results globally suggest that when they are properly activated, antigen-unspecific B-lymphocytes can present exogenous antigens by MHC class II molecules and stimulate peripheral antigen-specific T cells. Antigen presentation by activated B cells could be exploited for immunotherapy by allowing the in vitro generation of T cells specific against antigens expressed by tumors or viruses.