Intracellular cytokine staining is an increasingly popular analytical tool that can be used to define the profile of cytokines in various disease states. One important requirement for this assay is the inclusion of a protein transport inhibitor in stimulated cell cultures to trap the cytokine, thus allowing a brighter signal. Two compounds commonly used for this purpose are brefeldin A (BFA) and monensin (MN). Flow cytometry was used to assess the differential effects of BFA and MN on surface CD3, -4, -8, and -69 expression and the intracellular expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) following stimulation with phorbol myristate acetate and ionomycin. We found that BFA blocked the majority of CD3(+) cells from expressing surface CD69, but BFA did not inhibit intracellular CD69 expression. MN did not significantly inhibit surface CD69 expression. With regard to lymphocyte marker expression following activation, surface CD4 expression was significantly downregulated; however, less downregulation was observed with BFA treatment than with MN treatment. Analyzing intracellular cytokine expression, BFA trapped a greater percentage of TNF-alpha inside activated cells than MN. An analysis of the cytokine concentration in culture supernatants indicated that cells treated with MN released TNF-alpha and IFN-gamma from the cells, while the BFA-treated cells released IFN-gamma only. With prolonged (18-h) stimulation, the cells treated with MN were less viable than those treated with BFA. We conclude that the choice of a protein transport inhibitor is an important variable in this assay. When developing this method as a tool for clinical immunology laboratory analysis, investigators should consider the differential effects of BFA and MN on results.