Hepatitis B surface antigen (HBsAg)-specific T cells were analysed in 16 healthy individuals vaccinated against hepatitis B, using optimised protocols. HBsAg-specific IFN-gamma producing CD4(+) T cells were found at a similar low frequency (0.01%) within the naive T cell subset, the central and the effector memory T cell subset. Overall, HBsAg-specific total CD4(+) T cells were detected in approximately 81% of the HBV vaccinees. The use of dendritic cells substantially increased the otherwise low proliferative responses but not the percentage of IFN-gamma producing cells. The analysis of readily detectable CMV-specific CD4(+) T cell responses was used as a positive control and confirmed the adequacy of our assays. T cell responses associated with (in-) adequate hepatitis B vaccination can now be analysed in more detail.