To explore the relative importance of direct presentation vs cross-priming in the induction of CTL responses to viruses and viral vectors, we generated a recombinant vaccinia vector, vUS11, expressing the human CMV (HCMV) protein US11. US11 dislocates most allelic forms of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum into the cytosol, where they are degraded by proteasomes. Expression of US11 dramatically decreased the presentation of viral Ag and CTL recognition of infected cells in vitro without significantly reducing total cell surface MHC class I levels. However, because US11 is an endoplasmic reticulum resident membrane protein, it cannot block presentation by non-infected cells that take up Ag through the cross-priming pathway. We show that the expression of US11 strongly inhibits the induction of primary CD8+ CTLs when the infection occurs via the ip or iv route, demonstrating that direct priming is critical for the induction of CTL responses to viral infections introduced via these routes. This effect is less dramatic following im infection and is minimal after sc or intradermal infection. Thus, classic MHC class I Ag presentation and cross-priming contribute differentially to the induction of CD8+ CTLs following exposure to vaccinia virus via different routes.