The measurement of cytokines produced by activated T cells refines assessment of cellular immune function and facilitates whole blood T cell assays. The latter approximate conditions in vivo and obviate the need to purify blood mononuclear cells. We have investigated the parameters of the whole blood assay in humans to standardize and optimize the detection of tetanus-specific T cell cytokine responses. Optimal conditions include the use of undiluted whole blood, an incubation time of 36-48 h and a minimum of delay between venesection and incubation of the blood with antigen. Blood should be drawn at a standard time of day to minimize inter-assay variation due to diurnal rhythmicity in cytokine production. Interferon-gamma or interleukin-2 are specific and reliable readouts; other cytokines can be measured to further characterize the TH1 and TH2 elements of the T cell responses, although tetanus-stimulated IL-4 production is detected in only a minority of healthy individuals. The whole blood assay is a potentially valuable tool for assessing cellular immune function and screening for antigen-reactive T cells in humans.