Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgamma Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgamma is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNgamma(+) labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNgamma-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNgamma-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3(+)CD56(+) and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60-70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNgamma secretion. These results clearly demonstrate that the IFNgamma(+) subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNgamma(+) secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.