To assess the role of CC-chemokine ligand 5 (CCL5)/RANTES in opiate drug abuse and human immunodeficiency virus type 1 (HIV-1) comorbidity, the effects of systemic morphine and intrastriatal HIV-1 Tat on macrophage/microglial and astroglial activation were assessed in wild-type and CCL5 knockout mice. Mice were injected intrastriatally with vehicle or Tat and assessed after 7 days. Morphine was administered to some Tat-injected mice via time-release implant (5 mg/day, s.c. for 5 days) starting at 2 days post injection. Glial activation was significantly reduced in CCL5(-/-) compared to wild-type mice at 7 days following combined Tat and morphine exposure. Moreover, the percentage of 3-nitrotyrosine immunopositive macrophages/microglia was markedly reduced in CCL5(-/-) mice injected with Tat +/- morphine compared to wild-type counterparts, suggesting that CCL5 contributes to nitrosative stress in HIV-1 encephalitis. In CCL5(-/-) mice, the reductions in Tat +/- morphine-induced gliosis coincided with significant declines in the proportion of CCL2/MCP-1-immunoreactive astrocytes and macrophages/microglia compared to wild-type counterparts. In knockout mice, neither Tat alone nor in combination with morphine increased the proportion of CCL2-immunoreactive astrocytes above percentages seen in vehicle-injected controls. Macrophages/microglia differed showing modest, albeit significant, increases in the proportion of CCL2-positive cells with combined Tat and morphine exposure, suggesting that CCL5 preferentially affects CCL2 expression by astroglia. Thus, CCL5 mediates glial activation caused by Tat and morphine, thereby aggravating HIV-1 neuropathogenesis in opiate abusers and non-abusers. CCL5 is implicated as mediating the cytokine-driven amplification of CCL2 production by astrocytes and resultant macrophage/microglial recruitment and activation.