Abstract Feline immunodeficiency virus (FIV) induces neurological abnormalities in domestic cats. Previously, we demonstrated that two disparate strains of FIV (FIV-34TF10 and FIV-PPR) varied greatly in the ability to replicate in feline cortical astrocytes. To investigate the impact of the env region on the replication efficiency of these strains, we constructed two env chimera viruses, FIV-34TF10-PPRenv and FIV-PPR-34TF10env, to infect feline cortical astrocytes in vitro. Although all of these viruses infected cortical astrocytes, the efficiency of replication depended on strain, and the env region played an essential role. The viruses containing the env of 34TF10, FIV-34TF10, and FIV-PPR-34TF10env had the greatest replication rate, whereas the viruses containing the env of PPR replicated at a lower level. Other viral regions had modulatory effects on the replication rate, with the FIV-PPR genome providing a slight replication advantage over the FIV-34TF10 genome. We also monitored the effects of these viruses on an important astrocyte function, glutamate uptake; all viruses significantly decreased this activity, but only the viruses containing the env of PPR significantly impaired glutamate uptake without altering the culture viability. These results may be particularly relevant in the context of lentivirus-induced central nervous system disease in which a selective breakdown of astroglial function may contribute to neurodegeneration.