BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects. The majority of these individuals possessed populations of both CD8⁺ and CD4⁺ T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4⁺ T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-γ and tumor necrosis factor (TNF)-α, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4⁺ T lymphocytes. Secretion of both TNF-α and IFN-γ by CD154⁺CD4⁺ T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154⁺CD4⁺ T cells producing only IFN-γ or neither cytokine. These healthy subjects also harbored populations of functional CD8⁺ T cells specific for one or more of three newly defined HLA-A*02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A*02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4⁺ T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.