Highly enriched CD4+ and CD8+ human T cells were obtained from peripheral blood using a relatively simple and inexpensive method consisting of four steps: separation of mononuclear cells on Lymphoprep, removal of adherent monocytes by incubation in plastic petri dishes, removal of B cells, NK cells and further depletion of nonadherent monocytes by panning with anti-CD19, -CD16, -CD14, -CD11b and -CD33 mAb, and separation of CD4+ and CD8+ T lymphocytes by magnetic cell sorting (MACS). Cell culture for up to 48 h showed preservation of function by both positively and negatively selected cells as determined by production of IL-8. Although the cell separation procedure had no effect on interleukin-2 receptor (IL-2R, CD25) expression, it induced production of IL-4 by both T cell subsets selected positively, implying cell activation by ligation of CD4 and CD8 molecules. Irrespective of the mode of separation, CD8+ T cells produced more IL-4, a cytokine which is associated with a Th2-type cytokine profile of CD4+ T cells. We conclude that our method for separating T cells into their CD4+ and CD8+ subsets results in high cell purities with preservation of function, as determined by cytokine generation. If enriched cells are to be used for functional studies we recommend isolation by negative selection which has less effect on cell function. The relevance of the finding that CD8+ T cells can be an important source of IL-4 remains to be elucidated.