Considerable controversy exists over whether astrocytes can support human immunodeficiency virus (HIV) infection. We evaluated the impact of three cytokines critical to the development of HIV neuropathogenesis, gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha, on priming astrocytes for HIV infection. We demonstrate that IFN-gamma was the most potent in its ability to facilitate substantial productive HIV infection of an astroglioma cell line (U87MG) and human fetal astrocytes (HFA). The mechanism of IFN-gamma-mediated priming of HIV in HFA is unlikely to be at the level of up-regulation of receptors and coreceptors relevant to HIV entry. These data demonstrate that cytokine priming can alter HIV replication in astrocytes.