The structure and single-stranded (ss) RNA-binding by the nonstructural protein NS2 of three different orbiviruses were studied and compared. African horsesickness virus (AHSV), bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) were analyzed in recombinant baculovirus-infected cells and in cells infected with BTV and AHSV. Sedimentation analysis and nonreducing SDS-PAGE revealed that NS2 of all three orbiviruses is a 7S multimer with both inter- and intramolecular disulfide bonds, probably consisting of six or more NS2 molecules. The 7S NS2 multimer of all three viruses binds ssRNA but there is a marked disparity in the ssRNA-binding ability between the three proteins. At physiological salt concentration, BTV NS2 binds ssRNA very efficiently, whereas AHSV NS2 shows only a low efficiency for binding ssRNA. EHDV NS2 binds with intermediate efficiency. The result was the same irrespective of whether poly(U)-Sepharose or viral mRNA was used, indicating that ssRNA-binding by NS2 is nonspecific. The difference in RNA-binding ability may be related to the alpha-helix content of the respective proteins. NS2 of BTV has the highest predicted alpha-helix content followed by EHDV and AHSV. The ability of the NS2 proteins to form virus inclusion body-like structures in baculovirus-infected cells is not affected by the ssRNA-binding disparity.