Feline immunodeficiency virus (FIV) infection induces syncytium formation and cell death in primary feline astrocyte cultures but persistently and noncytopathically infects Crandell feline kidney cells (CrFK). Because viral envelope glycoproteins are implicated in cell fusion events we evaluated the astrocyte-produced FIV surface glycoprotein for properties that might distinguish it from that produced in CrFK cells. The surface glycoprotein from astrocytes migrated faster on SDS-PAGE and contained more Endo H-sensitive oligosaccharides than that from CrFK, although the precursor and deglycosylated envelope glycoproteins from both cells were the same size. Castanospermine treatment of infected astrocytes, which blocks glucose trimming from oligosaccharide side chains of glycoproteins, both obliterated the mobility difference between astrocyte- and CrFK-produced FIV surface glycoproteins and prevented syncytium in infected astrocyte cultures. These results demonstrate the importance of the infected cell type in viral envelope protein glycosylation and implicate cell type-specific carbohydrate structures on retroviral glycoproteins as mediators of cell fusion.