A small percentage of astrocytes are consistently infected in vivo by HIV-1 and may contribute to neuropathogenesis despite a non-productive infection. Overexpression of the nef gene product has been associated with their infection both in vivo and in vitro. We examined the role of the nef gene during HIV replication in astrocytes (U251MG cells) following transfection with pNL4-3 proviral plasmid or isogenic strains containing a deletion or point mutation in the nef gene (pNL4-3deltaNef; pNL4-3-nef-stop). We were able to initiate virus replication which peaked at 5 days post-transfection and became non-productive after 21 days. Nef protein expression by wild type pNL4-3 was observed at low levels compared to control HeLa cells at peak virus replication. At later time points after development of a non-productive infection, viral antigen and Nef protein was not detectable however virus was readily recovered by co-culture with CD4+T-cells. Interestingly, virus production was significantly enhanced by a 222 base pair deletion in the nef reading frame. This was not observed with a frame shifting point mutation in nef, indicating a suppressive effect of nef on virus production in astrocytes. The enhanced virus production from nef-deleted pNL4-3 in U251MG cells was not reversed by co-expression of Nef from a second Nef-expressing plasmid, and in fact Nef expression in trans had a further positive effect on virus production. This suggested opposing effects of the Nef protein and elements contained within the nef sequence on virus production in astrocytes. Despite the low expression of Nef by U251MG astrocytes, relatively high amounts of multiply spliced 2 kb mRNA were present compared to HeLa cells. These data demonstrate that an acute low-level infection of astrocytes rapidly becomes a non-productive infection and this process is assisted by sequences in nef. The low level Nef protein expression, despite high levels of mRNA, suggests a block in translation of multiply spliced HIV mRNA in astrocytes, or a translational control mechanism not yet characterised.