Interleukin 4 (IL-4) and interferon gamma (IFN-gamma) production was analysed in murine spleen cells during primary and secondary mitogen stimulation in vitro. The kinetics, frequency and phenotype of single lymphokine-producing cells were studied by combining intracytoplasmatic immunofluorescence and surface staining. Both IL-4 and IFN-gamma was produced by CD4+ as well as CD8+ cells, however 75-80% of IL-4 producers were CD4+ and 90% of IFN-gamma+ cells were CD8+. In primary stimulations, concanavalin A (Con A) activation or anti-CD3 antibody together with phorbol 12-myristate 13-acetate (PMA) induced different patterns of lymphokine production. Approximately the same frequency of IFN-gamma+ cells was induced by both stimulation procedures but the kinetics was different with a peak at 30 h using Con A and at 52 h using anti-CD3 and PMA. IL-4 production peaked at 52 h, but the frequency of IL-4+ cells was 8-10 times higher after stimulation by anti-CD3 and PMA than after Con A stimulation. During restimulation of the mitogen activated cells, lymphokines were rapidly produced; both IL-4 and IFN-gamma production peaked at 8-11 h. Only a small increase in the frequency of IL-4+ cells was seen, at most two to three times. No evidence for a major shift of lymphokines produced between primary and secondary stimulations could be found. Instead, the pattern of lymphokine production induced by the primary stimulus was dominant also in secondary cultures irrespective of stimulation condition.