Assessment of T cell activation has traditionally been performed by measuring proliferation as a function of 3[H]-thymidine incorporation, or secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture. An alternative method for detection of proliferation at the single cell level utilizes incorporation of bromodeoxyuridine (BrdU), an analog of thymidine, into cellular DNA. After appropriate fixation and permeabilization of the cells, a monoclonal antibody (mAb) against BrdU conjugated with a fluorescent dye is employed to measure by flow cytometry the incorporated BrdU. Here, we report a flow cytometric procedure which can be used for the simultaneous detection of BrdU incorporation, activation markers such as CD69 and CD25, and intracellular cytokines in T cell subsets from activated PBMC. Our observations are consistent with the proposal that cytokine synthesis and cell proliferation occur sequentially in CD4+ T cells stimulated with the superantigen staphylococcal enterotoxin B (SEB). The majority of cells expressing the cytokines IFN-gamma and IL-2 at 48 h appear to have undergone DNA synthesis, however all proliferating cells do not express IFN-gamma or IL-2. The methods presented in this report offer a unique approach for studying simultaneous expression of key cellular activation events in phenotypically resolved lymphocyte populations.