Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus 27 within family Picornaviridae. To investigate target proteins recognised by T-cells in human enterovirus B infections, viral-encoded structural (VP0 [VP4 and VP2], VP1, VP3) and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in E. coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognised enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of 19 (79%) donor lines. Non-structural 2C protein was recognised in 11 of 19 (58%) lines, and 11 of 19 (58%) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.