The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II+CD11c− non-T, non-B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II+CD11c− non-T, non-B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag-2−/− mice with adoptively transferred normal spleen cells indicated that these cells were non-lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II+CD11c− non-T, non-B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin-6 in response to infected red blood cells, but had only a limited ability to activate antigen-specific CD4+ T cells. This study revealed a novel interaction between MHC II+CD11c− non-lymphoid cells and lymphoid cells in the accumulations of these non-lymphoid cells in the spleen during infection with P. yoelii.