We investigated the function of human double-degative T lymphocytes (CD4–CD8–CD3+) in 8 adult volunteers. Using Rosettesep-human T cell, CD3+ T lymphocytes were negatively selected and further stimulated with 1-, 3-phorbor myristate acetate and ionomycin in the presence of brefeldin A for 4 hours. Thereafter, these cells were stained for surface CD4 and CD8, and intracellular interferon-g and interleukin-4. The results indicate that CD4+ T lymphocytes, CD8+ T lymphocytes and double negative T cells show the similar function of cytokine production.