Invasive aspergillosis (IA) is a major cause of infection-related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11-aa overlap with previous peptide) spanning the entire 427-aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1-type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4(+) T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA-B3501(+) donors were found to be strongly cytotoxic to autologous Asp f16-peptide pool- and Aspergillus culture extract-pulsed targets after 4-5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)-gamma production were mediated exclusively by CD8(+) T cells in response to pool-pulsed targets. Interleukin (IL)-4 production was not detected. CTL activity was restricted by HLA-B3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool-pulsed B3503(+) BLCL but not B3502(+) or B3508(+) BLCL presented peptide to donor no. 1. B3503(+) BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA-Class I-restricted, Aspergillus-specific T cells that may be capable of conferring immunity to IA.