We have reported that Listeria monocytogenes-specific IFN-gamma-producing CD4+ T cells are induced by in vitro 5-day culture of naive spleen cells with viable L. monocytogenes (VLM), but not induced by culture with heat-killed L. monocytogenes (HKLM). In the present study, the role of B cells in the regulation of induction of IFN-gamma-producing CD4+ T cells in the in vitro system was investigated. We found that L. monocytogenes-specific IFN-gamma-producing CD4+ T cells were not generated when B cells were depleted from spleen cells before culture with VLM although Ag-specific proliferative response was retained. IFN-gamma production by CD4+ T cells was restored by addition of B cells cultured with VLM to the culture of B cell-depleted spleen cells and VLM. In contrast, B cells cultured with HKLM could not restore the induction of IFN-gamma production when added in culture of B cell-depleted spleen cells and VLM. Analysis of cytokine gene expression by reverse transcription-polymerase chain reaction method revealed that expression of interleukin-10 (IL-10) was higher but TNF-alpha was lower in B cells cultured with HKLM when compared with that in B cells cultured with VLM. Furthermore, addition of neutralizing anti-IL-10 mAb into culture of naive spleen cells with HKLM resulted in appearance of IFN-gamma-producing cells. These results suggest that B cells have positive and negative roles in the induction of IFN-gamma-producing CD4+ T cells. The inhibition of induction of IFN-gamma-producing CD4+ T cells may depend on B cell-derived IL-10.