The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8(+) T cell populations specific for the HLA-A*0101 restricted epitope NP(44-52) and the HLA-A*0201 restricted epitope M1(58-66) were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-gamma and tumour necrosis factor-(TNF)-alpha upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-gamma and TNF-alpha by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm(+) and M1-Tm(+) cells differed considerably. Overall, no difference in the proportion of NP-Tm(+) or M1-Tm(+) cells expressing CD107a was observed. The proportion of M1-Tm(+) cells that produced IFN-gamma (P < 0.05) was larger than for NP-Tm(+) cells, independent of IL-2 concentration. When cultured under IL-2(hi) concentrations higher TNF-alpha expression was also observed in M1-Tm(+) cells (P < 0.05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm(+) and NP-Tm(+) cells.