Dysregulation of cytokines and chemokines during human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV) infection is thought to be critical in the progression of acquired immunodeficiency syndrome (AIDS). To evaluate the potential role of Th1-agonist chemokines in disease progression during AIDS, we assessed CXCL9/MIG and CXCL10/IP-10 expression simultaneously in the periphery and lymphoid tissues of SIV-infected animals at a single-cell level by flow cytometry. We optimized intracellular staining and analysis of CXCL9/MIG and CXCL10/IP-10 production in human leukocyte antigen (HLA)-DR+ macaque cells by flow cytometry using cross-reactive antibodies against human chemokines. We observed an upregulation of CXCL9/MIG and CXCL10/IP-10 production in both the periphery and lymph nodes of infected animals compared with naïve controls. Animals with higher viral loads had higher levels of CXCL9/MIG and CXCL10/IP-10 producing cells compared with animals with low viral loads. Analysis of cells bearing the receptor (CXCR3) for CXCL9/MIG and CXCL10/IP-10 revealed increased number of CXCR3+ cells in the lymph nodes of infected animals. Importantly, an inverse correlation (P < 0.05) between CXCL9/MIG and CXCL10/IP-10 production, both in the periphery and lymph nodes, and peripheral CD4+ T-cell numbers was observed. These findings provide further evidence that dysregulation of Th1 agonist chemokines might contribute to the ultimate immunopathology during AIDS.