In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80% of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the cross-reactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.