A number of studies suggest that cytokines may contribute to the pathogenesis of viral infections, including dengue. In this study, we developed a double immunocytochemical method and characterized cytokine-producing cells in the peripheral blood mononuclear cells (PBMC) of dengue virus-immune donors after in vitro stimulation with specific dengue antigens. We found that double immunostaining using immunoalkaline phosphatase (Vector blue) for cytokines [interferon-gamma (IFN-gamma), interleukin (IL) -2, -4, -1alpha, -1beta, and -6, tumor necrosis factor beta (TNF-beta), and TNF-alpha] and immunoperoxidase [diaminobenzidine (DAB)] for cell surface markers (CD3, CD4, CD8, CD20, and CD68) provided the best distinction of double-positive cells from single-positive or -negative cells. The number of IFN-gamma, IL-2, IL-4, and TNF-beta-positive cells increased 2 or 3 days after stimulation with specific dengue antigens. No or very few cytokine-producing cells were detected in the PBMC of non-immune donors stimulated with dengue antigens and the PBMC of immune donors stimulated with a control antigen. The analysis of cell surface markers showed that mainly CD4+ and CD8+ T cells produced these cytokines. The results obtained by immunocytochemistry were consistent with cytokine levels detected in the culture medium assayed by enzyme-linked immunosorbent assay. In conclusion, this double immunocytochemistry technique is suitable for the detection and characterization of cytokine-producing cells in PBMC. Furthermore, the results support the hypothesis that antigen-stimulated CD4+ and CD8+ T cells produce cytokines that may play a role in the pathogenesis of dengue virus infection.