The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor a (TNFa) at the cell surface through the use of a specific inhibitor of the TNFa-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFa and makes it possible to assess, in the same measurement, the phenotype of TNFa þ -secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFa þ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFa expression on the majority of CD3 þ T cells and secretion of Th1-associated cytokines: interferon c, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14 þ monocytes showed surface TNFa expression; in parallel, high amounts of soluble IL1b, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viralspecific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFa þ monocytes, TNFa þ /CD8 þ T cells and TNFa þ /CD8 À T lymphocytes in association with an increased secretion of IFNc, IL6 and TNFa.