Screening with the flow cytometric IFN-γ assay has led to the identification of a new immunogenic peptide (SSYRRVPGI) from the influenza PB1 polymerase (PB1 703–711) and a mimotope (ISPLMVAYM) from the PB2 polymerase (PB2 198–206). CD8+ T cells specific for K b PB1 703 make both IFN-γ and TNF-α following stimulation with both peptides. The CD8+ K b PB1 703+ population kills PB2 198-pulsed targets, but cell lines stimulated with PB2 198 neither bind the K b PB1 703 tetramer nor become CTL. This CD8+ K b PB1 703+ population is prominent in the primary response to an H3N2 virus, although it is much less obvious following secondary challenge of H1N1-primed mice. Even so, we can now account for> 40% of the CD8+ T cells in a primary influenza pneumonia and> 85% of those present after H3N2→ H1N1 challenge. Profiles of IFN-γ and TNF-α staining following in vitro stimulation have been traced for the four most prominent influenza peptides through primary and secondary responses into long-term memory. The D b NP 366 epitope that is immunodominant after the H3N2→ H1N1 challenge shows the lowest frequencies of CD8+ IFN-γ+ TNF-α+ cells for> 6 wk, and the intensity of IFN-γ staining is also low for the first 3 wk. By 11 wk, however, the IFN-γ/TNF-α profiles look to be similar for all four epitopes. At least by the criterion of cytokine production, there is considerable epitope-related functional diversity in the influenza virus-specific CD8+ T cell response. The results for the K b PB1 703 epitope and the PB2 198 mimotope also provide a cautionary tale for those using the cytokine staining approach to identity antigenic peptides.