Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.