Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.