Recent in vitro evidence suggests two alternative mechanisms by which bone marrow–derived APCs may process exogenous antigens for presentation to CTL in vivo, a phenomenon termed cross-priming. Although in vitro studies have suggested that both TAP-dependent and TAP-independent pathways exist, we have now demonstrated an absolute requirement for a functional TAP for cross-priming to occur in vivo. Bone marrow chimeras reconstituted with marrow from TAP-defective donors develop functional CD8⁺ CTL, but have APCs with disrupted TAP function. In such chimeras, in vivo priming of naive CTL was observed when antigen was targeted to the ER in a TAP-independent fashion, but cross-priming could not be demonstrated. These results support the TAP-dependent mechanism of cross-priming.