In this paper, the relative efficacy of macrophages, B cells and splenic dendritic cells (SDC) in presenting West Nile virus (WNV) antigens to WNV memory CD4+ T cells is examined. The results indicate that, under appropriate conditions, all these cell types can function as antigen-presenting cells (APC). Listeria-induced peritoneal macrophages induced higher proliferative responses than SDC or B cells derived from naive or 14 day WNV-primed mice. The ability of Listeria-induced macrophage populations to present antigen was specifically inhibited by anti-Class II major histocompatibility complex (MHC) antibodies. On a cell population basis, B cells obtained from mice primed with WNV 14 days previously evoked higher responses than resting B cells. B cells from mice receiving weekly injections of WNV over a period of 4 weeks elicited optimal responses with lower doses of antigen than naive or 14 day WNV-primed B cells. When macrophages were used as APC, addition of specific antibodies to WNV resulted in increased efficiency of presentation, probably due to increased uptake of antigen by opsonization. In contrast, addition of anti-WNV antibodies to hyperimmune B cells reduced their efficacy presumably by reducing uptake of antigen by B cell surface immunoglobulin. When SDC from C57BL/6 mice were used as APC, WNV-specific proliferative responses were directly related to the number of stimulator cells used, and the background proliferation with mock antigen was two- to five-fold lower than specific responses. Higher levels of background proliferation were stimulated by SDC from CBA/H mice so that the antigen-specific responses were always less than two-fold higher than background.