HIV-1 invades the CNS and productively infects macrophages and microglial cells. Both cell types express CD4 and the necessary coreceptors (CCR5 and CXCR4) for HIV entry. Astrocytes are also infected with HIV-1 however although they do while they express CCR5 and CXCR4 they lack expression of CD4. The mechanism by which HIV enters astrocytes remains unclear but is thought to involve endocytic vesicle formation, the nature of which is unknown. The fate of HIV-1 virions following entry into astrocytes is also unclear. While tWhile there is evidence that HIV proviral DNA can become integrated into the astrocyte genome, the ability of astrocytes to transmit the virus to susceptible cells, thus contributing to spread within the CNS has been suggested. To track HIV entry into astrocytes, we have developed stringent methodology to tag HIV particles with two fluorescent proteins. The first fluorescent tag, eGFP, is encoded within the HIV structural protein Gag and is flanked by HIV protease cleave sites. This latter feature results in eGFP becoming cleaved from Gag and acting a soluble green “content” marker within the virion. The second tag is achieved by fusing a well-known HIV core protein, Vpr, directly to the red fluorescent protein mCherry. By using two tags, we can visualize in real time trafficking of intact virions (yellow-double positive particles) and also virions that have fused with the plasma membrane (single red core positive particles). Using fluorescent real-time microscopy,Utilizing this approach we have tracked EGFP-labeledfluorescently labeled HIV viral particles as they infectfate in astrocytes. By simultaneouslySimultaneous staining for different vesicular compartments we havehas identified potential receptors associated with vesicle formation. Additionally we have demonstrated that, in addition to becoming infected, astrocytes can efficiently transmit intact infectious virus to susceptible cells. Infection of astrocytes is pivotal to the development of HIV associated neurocognitive disorders. associated with HIV. Understanding the mechanism by which HIV enters astrocytes is important for developing strategies aimed at prevention and eradication of HIV.