Exogenous Ag in the extracellular fluids do not gain access to the class I Ag-presenting pathway in most cells. However, there is an APC resident in spleen that can process and present exogenous Ag in association with class I molecules. We characterize the phenotype of this cell. This APC is of low buoyant density, is adherent to Sepharose and glass, and expresses both class II molecules and FcR. This phenotype identifies this APC as a macrophage. Resident, peptone- and thioglycolate-induced peritoneal macrophages also display this Ag-presenting activity. Analysis with CTL clones suggest that this Ag-presenting pathway may be active in only a subset of macrophages. A similar Ag-presenting activity is also present in dendritic cell-enriched populations from spleen although we cannot rule out the possible involvement of contaminating macrophages. In contrast, B and T cells that are resident in spleen and LPS blasts are unable to present exogenous Ag in association with class I molecules. The presentation of exogenous OVA with class I molecules is not inhibited by the inhibitors of thiol proteases, leupeptin, and antipain. The presence of gelonin, a ribosomal inactivating protein, in the extracellular fluids inhibits the ability of these APC to present exogenous OVA. Under identical conditions, gelonin does not inhibit Con A-stimulated T cell proliferation, or LPS-stimulated B cell proliferation and Ag presentation. These results are discussed in relation to the potential pathways through which an Ag in the extracellular fluids is presented with MHC class I molecules.