Dendritic cells (DCs) use cellular pathways collectively referred to as cross-presentation to stimulate CD8(+) T cells with peptide Ags derived from internalized, exogenous Ags. We have recently reported that DCs rely on aminoterminal trimming of cross-presented peptides by insulin-responsive aminopeptidase (IRAP), an enzyme localized in a regulated endosomal storage compartment. Considering a report contending that this role is limited to inflammatory DCs (Segura et al. 2009. Proc. Natl. Acad. Sci. USA 106: 20377-20381), in this study, we examined the role of IRAP in steady-state DC subpopulations. Steady-state conventional DCs (cDCs) and plasmacytoid DCs expressed similar amounts of IRAP. IRAP colocalized with the endosomal markers Rab14 and syntaxin 6, both known to be associated with regulated endosomal storage compartments, in CD8(+) and CD8(-) cDCs-however, to a greater extent in the former population. Likewise, IRAP recruitment to phagosomes was significantly stronger in CD8(+) DCs. IRAP deficiency compromised cross-presentation of soluble and particulate Ag by both CD8(+) and CD8(-) cDCs, again with a stronger effect in the former population. Thus, the requirement of IRAP in cross-presentation extends to steady-state cDCs. Moreover, these data suggest that increased recruitment of an IRAP(+)/Rab14(+) compartment to Ag-containing vesicles contributes to the superior cross-presentation efficacy of CD8(+) cDCs.