Novel antigen presenting cells (APCs) have been described in the murine spleen. Cells have a distinct CD11c(lo)CD11b(hi)MHC-II(-)CD8α(-) phenotype as highly endocytic dendritic-like cells that cross-present antigen to CD8(+) T cells but fail to activate CD4(+) T cells. These cells are named "L-DCs" because they reflect dendritic cells (DCs) produced in long-term spleen cultures (LTC). Similar cells were produced when bone marrow progenitors were cocultured over the splenic stromal line 5G3. Cocultures continuously produced a majority of L-DCs and a transient population of cells reflecting conventional dendritic cells (cDCs). Both the L-DC and cDC-like subsets cross-present antigen to CD8(+) T cells, inducing their activation and proliferation. However, as MHC-II(-) cells, L-DCs are unable to activate CD4(+) T cells, while MHC-II(+) cDC-like cells present antigen for CD4(+) T cell activation. These results distinguish two APC subsets produced in vitro: a transient population of cDC-like cells and L-DCs that are continuously produced, presumably from self-renewing progenitors. These subsets are not developmentally linked via a precursor or progeny relationship. L-DCs and cDC-like cells are also distinct in terms of cytokine expression, with 65 of 84 tested genes displaying greater than a twofold difference by quantitative reverse-transcriptase polymerase chain reaction. Splenic stroma supports production of two APC subsets reflecting different lineage origins.