Chemokine receptors and their ligands play a prominent role in regulating leucocyte migration. In the local milieu of inflammation, a high concentration of chemokines can recruit different chemokine receptor-expressing lymphocytes. To understand the distinct immunological functions of CXC chemokine receptor 3 (CXCR3+) and CC chemokine receptor 4 (CCR4+) cluster of differentiation 4 (CD4+) T-cells accumulated in human tuberculosis (TB) pleural fluid after tuberculous antigen stimulation. Mononuclear cells were isolated from the peripheral blood of healthy donors, cord blood and TB pleural fluid, and expression of CXCR3 and CC chemokine receptor 4 (CCR4), cytokines and cytolytic molecules by CD4+ T-cells with or without stimulation were analysed using fluorescence-activated cell sorting. CXCR3 and CCR4 expression on CD4+ T-cells from pleural fluid mononuclear cells (PFMCs) was significantly higher than in peripheral blood mononuclear cells (PBMCs). T-cell receptor signalling resulted in the upregulation of CXCR3 and CCR4 expression on CD4+ T-cells from cord blood mononuclear cells (CBMCs) and PBMCs in a time-dependent manner, but not from PFMCs. After stimulation with Mycobacterium tuberculosis antigens, CXCR3+CCR4-CD4+ T-cells were dominated by multifunctional T-helper 1 cells; however, CXCR3+CCR4+CD4+ T-cells exhibited cytotoxicity and degranulation by expressing granzyme B, perforin, CD107a/b and tumour necrosis factor-related apoptosis-inducing ligand. Our results indicated that CXCR3 or CCR4 expression on CD4+ T-cells had different biological activities against tuberculous infection, and could be a potential marker for the diagnosis of TB.