Polyomavirus BK (BKV) is known as an important etiologic agent in the development of hemorrhagic cystitis (HC) after allogeneic stem cell transplant (SCT). To define T cell epitopes of the BKV proteins VP1 and sT, eight potential HLA-A2-binding peptides were synthesized based on computer algorithms. These peptides were co-incubated with CD8 + T cells from the peripheral blood (PB) of 25 healthy volunteers and seven patients suffering from HC after allogeneic SCT in a mixed-lymphocyte peptide culture (MLPC), which were subsequently screened by enzyme-linked immunospot (ELISPOT) assays and fluorescence-activated cell sorting (FACS) analysis. We found that CD8 + T cells from five of seven (71%) patients with HC presensitized with the BKV peptide VP1 p108 (LLMWEAVTV) specifically recognized T2 cells pulsed with VP1 p108. In contrast, only seven of 25 (28%) healthy volunteers had CD8 + T cells reactive with VP1 p108-pulsed T2 cells. The presence of VP1 p108-specific T cells could be confirmed by FACS analysis. The BKV peptide VP1 p108 seems to play an important role as an immunodominant peptide in the pathogenesis of HC in patients after allogeneic SCT, and might be a promising target for immunotherapies or even strategies to prevent the development of BKV-associated HC.