Antiviral or antitumor immunity requires activation of cytotoxic CD8⁺ T cells by dendritic cells, which present viral or tumor antigens on major histocompatibility complex (MHC) class I molecules. The intracellular mechanisms facilitating MHC class I–restricted presentation of extracellular antigens ('cross-presentation') are unclear. Here we demonstrate that cross-presentation of soluble antigen occurred in an early endosomal compartment distinct from the endoplasmic reticulum where endogenous antigen is loaded onto MHC class I. Efficient cross-presentation required endotoxin-induced, Toll-like receptor 4– and signaling molecule MyD88–dependent relocation of the transporter associated with antigen processing, essential for loading of MHC class I, to early endosomes. Transport of cross-presented antigen from endosomes to the cell surface was inhibited by primaquine, which blocks endosomal trafficking. Thus, cross-presentation is spatially and mechanistically separated from endogenous MHC class I–restricted antigen presentation and is biased toward antigens containing microbial molecular patterns.