To assess the role of the phosphatase and tensin homologue on chromosome 10 (PTEN) in mediating envelope glycoprotein 120 (gp120)-induced neurotoxicity in the striatum, PTEN was silenced using short interfering RNA (siRNA) vectors. PTEN activity directs multiple downstream pathways implicated in gp120-induced neuronal injury and death. PTEN is a negative regulator of Akt (protein kinase B) phosphorylation, but has also been shown to directly activate extrasynaptic NMDA receptors and dephosphorylate focal adhesion kinase. Rodent striatal neurons were nucleofected with green fluorescent protein (GFP)-expressing siRNA constructs to silence PTEN (PTENsi-GFP) or with negative-control (NCsi-GFP) vectors, and exposed to HIV-1 gp120(IIIB) using rigorously controlled, cell culture conditions including computerized time-lapse microscopy to track the fate of individual neurons following gp120 exposure. Immunofluorescence labeling showed that subpopulations of striatal neurons possess CXCR4 and CCR5 co-receptor immunoreactivity and that gp120(IIIB) was intrinsically neurotoxic to isolated striatal neurons. Importantly, PTENsi-GFP, but not control NCsi-GFP, constructs markedly decreased PTEN mRNA and protein levels and significantly attenuated gp120-induced death. These findings implicate PTEN as a critical factor in mediating the direct neurotoxic effects of HIV-1 gp120, and suggest that effectors downstream of PTEN such as Akt or other targets are potentially affected. The selective abatement of PTEN activity in neurons may represent a potential therapeutic strategy for the CNS complications of HIV-1.