Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-γ secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14 days, we obtained an average of 1.1 × 10⁷ cells from a single 100-mL blood draw in 7 of 7 healthy individuals. Within another 14 days, these cells were expanded to an average number of 2.0 × 10⁸ T-helper 1 (T_H1) cells secreting IFN-γ on stimulation with Aspergillus antigens. Testing various fungal antigen extracts, similar proportions of IFN-γ-producing CD3⁺/CD4⁺ cells were obtained upon activation with antigen extracts of A fumigatus, A flavus, A niger, and Penicillium chrysogenum, whereas no significant IFN-γ production was observed upon activation with antigen extracts of Alternaria alternata and Candida albicans. In addition, generated T cells were able to induce damage to A fumigatus hyphae, and significantly increased hyphal damage induced by human neutrophils. CD4⁺ T-cell-mediated alloreactivity of generated anti-Aspergillus T cells was clearly reduced compared with that of the original cell population. In conclusion, we present a simple and feasible strategy for rapid generation of a high number of functional active T cells against Aspergillus from a single blood draw. Our data suggest that functionally active T cells against Aspergillus could be a promising treatment option for patients undergoing allogeneic SCT. (Blood. 2006;107: 2562-2569)