A CD8(+) cytolytic T-lymphocyte (CTL) response to antigen-presenting cells generally requires intracellular delivery or synthesis of antigens in order to access the major histocompatibility complex (MHC) class I processing and presentation pathway. To test the ability of pertussis toxin (PT) to deliver peptides to the class I pathway for CTL recognition, we constructed fusions of CTL epitope peptides with a genetically detoxified derivative of PT (PT9K/129G). Two sites on the A (S1) subunit of PT9K/129G tolerated the insertion of peptides, allowing efficient assembly and secretion of the holotoxin fusion by Bordetella pertussis. Target cells incubated with these fusion proteins were specifically lysed by CTLs in vitro, and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A, suggesting a dependence on intracellular trafficking events, but was not inhibited by the proteasome inhibitors lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Furthermore, the activity was present in mutant antigen-presenting cells lacking the transporter associated with antigen processing, which transports peptides from the cytosol to the endoplasmic reticulum for association with MHC class I molecules. PT may therefore bypass the proteasome-dependent cytosolic pathway for antigen presentation and deliver epitopes to class I molecules via an alternative route.