Monoclonal antibodies (MCAbs) were used to dissect the antigenic sites of the surface glycoproteins of the prototype cell-adapted Wyoming strain of equine infectious anemia virus (EIAV). Serologic reactivities of these MCAbs were determined by ELISA, additive ELISA, competitive ELISA, and Western blot assays. The results indicated that antigenic reactivity of gp90 was localized on at least four distinct epitopes, two of which were important in neutralization. Our studies also revealed that these epitopes were localized on overlapping antigenic sites on gp90. On the other hand, only two distinct non-overlapping epitopes were identified on gp45. Competitive binding studies of neutralizing MCAbs and reference EIA-positive horse serum delineated the presence of a neutralization domain on gp90 that appears to be immunodominant both in naturally infected horses and in mice immunized with EIAV. Limited proteolytic fragmentation of the gp90 component of several serologically distinct EIAV isolates produced common 12K immunoreactive fragments that contained a conserved epitope. These results indicate the occurrence of conserved antigenic regions on EIAV glycoproteins as well as a neutralization domain on gp90, which can be used as potential targets for vaccine development.