BACKGROUND Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. METHODS By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. RESULTS Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-γ- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-γ+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. CONCLUSIONS In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.