CD8+ cytolytic T lymphocytes (CTL) are almost certainly an important component of a potentially protective immune response to HIV. To test the ability of pertussis toxin (PT) to deliver an HIV-derived major histocompatibility complex (MHC) class I peptide for CTL stimulation, we constructed a fusion of the gp120 P18-I10 CTL epitope with a genetically detoxified derivative of PT (PT9K/129G) and assayed this fusion for its ability to stimulate a gp120-specific CTL response in vitro and in vivo. Antigen-presenting cells incubated with this fusion protein were lysed by P18-I10-specific CTL in vitro and this activity was shown to be MHC class I restricted. The activity was inhibited by brefeldin A but was not inhibited by proteasome inhibitors, possibly because PT undergoes retrograde intracellular transport through the Golgi apparatus to the endoplasmic reticulum and delivers epitopes directly to nascent class I molecules. Mice immunized intraperitoneally with a single dose of the fusion protein without adjuvant raised a strong gp120-specific CTL response in the spleen. This CTL response was dependent on (1) the dose of fusion administered, (2) the fusion of the epitope with the toxin (since coadministration of peptide and toxin gave no response), and (3) the activity of CD8+ cells. These data demonstrate that this detoxified derivative to PT, which is already a component of a licensed vaccine for humans, could represent a useful vaccine vector molecule for stimulation of HIV-specific CTL responses.