A new one-step culture-immunoassay procedure is described for testing cytokine production by immunocompetent cells in whole blood (WB) without the need for an isolation step. Briefly, WB samples or distilled water were added to RPMI medium containing specific anti-cytokine peroxidase-labelled monoclonal antibodies and incubated in micro-well plates coated with specific capture monoclonal antibodies, directed against distinct epitopes of the cytokine, and containing dried polyclonal activators (5.625 micrograms LPS + 1.125 micrograms PHA) or dried standards respectively. The optimalisation of the assay is described for an extended measurement range. The best compromise between sensitivity and linearity was obtained with the addition of 50 ng/well for TNF-alpha and IL-6 or 100 ng/well for IFN-gamma of unconjugate antibodies to the corresponding conjugate. The kinetics of individual production of each cytokine in WB of normal healthy donors showed values entering the standard range following incubation times of between 2 and 8 h for TNF-alpha, 2 and 4 h for IL-6, and 4 and 24 h for IFN-gamma. The sensitivity, the precision (intra-assay CVs) and the reproducibility (interassay CVs) of the assays were as follows: 70 pg/ml, < or = 14% and < or = 11% for TNF-alpha; 25 pg/ml, < or = 11% and < or = 16% for IL-6; 25 pg/ml, < or = 19% and < or = 20% for IFN-gamma. The accuracy (% of recovery) of the assays was in the order of 100% and between 40 and 60% in the absence or presence of polyclonal activators, reflecting the occurrence of an active production/consumption mechanism during the activation.