The identification and characterization of a functional cellular receptor for equine infectious anemia virus (EIAV), designated equine lentivirus receptor-1 (ELR1), a member of the tumour necrosis factor receptor protein family, has been reported previously [Zhang, B. et al. (2005). Proc Natl Acad Sci U S A, 102 , 9918-9923]. The finding of a single receptor for EIAV is distinct from feline, simian and human immunodeficiency viruses, which typically utilize two co-receptors for infection, but is similar to avian and murine oncoviruses, which use single receptors. This study sought to determine ELR1-binding domains of EIAV gp90. Towards this goal, a GFP-tagged gp90 fusion protein (gp90GFP) expression vector was constructed and a specific cell-cell binding assay was developed to measure EIAV gp90 binding to ELR1. Using these assays, the receptor-binding properties of 41 gp90GFP mutants were evaluated, each with a sequential replacement 11 aa linear epitope peptide from the vesicular stomatitis virus glycoprotein (VSV-G tag), as well as eight mutants containing individual gp90 variable-domain deletions. The results of these studies demonstrated that, in general, gp90 constructs containing substitutions or deletions in the N-terminal third of gp90 retained their receptor-binding activity. In contrast, segment substitutions or deletions in the C-terminal two-thirds of gp90 eliminated receptor-binding activity. Thus, these results reveal for the first time that the ELR1-binding domains of EIAV gp90 are located in the C-terminal two-thirds of EIAV gp90, apparently as a complex of discontinuous determinants.