Human immunodeficiency virus-1 (HIV-1) invades the brain early in infection and may cause HIV-associated dementia (HAD), which is characterized by reactive astrocytes, and macrophage and T-cell infiltrates. HIV-1 Tat protein is thought to contribute to HAD by transactivating host genes, such as that encoding monocyte chemoattractant protein-1 (MCP-1/CCL2), although its mechanisms of action are not fully understood. We investigated the molecular pathways involved in Tat-induced MCP-1/CCL2 gene expression in human astrocytes. We found that Tat induced MCP-1/CCL2 synthesis in human astrocytes infected with a lentivirus carrying the gene encoding Tat or treated with a biologically active synthetic Tat protein. The induction of MCP-1/CCL2 was independent of the nuclear factor kappaB (NF-kappaB) classical pathway, but was significantly inhibited by specific cyclin-dependent kinase 9 (cdk9) inhibitors, such as a dominant-negative mutant or siRNA. By contrast, broader-spectrum cdk inhibitors, such as roscovitine, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol, inhibited MCP-1/CCL2 induction by Tat. We also analyzed the effects of roscovitine, DRB, and flavopiridol on Tat-induced HIV-1 long terminal repeat (LTR) expression following the infection of astrocytes and HeLa cells. Astrocytes showed no inhibition by roscovitine, 59% inhibition by DRB, and 80% inhibition by flavopiridol. In control HeLa cells, high levels of inhibition were observed with roscovitine, DRB, and flavopiridol. We have ascertained the direct implication of cdk9 in Tat-induced MCP-1 expression by performing ChIP assay. These results demonstrate that cdk9 is involved in Tat-induced HIV-1 LTR, MCP-1/CCL2 gene expression.