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Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-γ (IFN-γ), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-γ were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals.