We have previously reported [Risatti, G.R., Borca, M.V., Kutish, G.F., Lu, Z., Holinka, L.G., French, R.A., Tulman, E.R., Rock, D.L. 2005a. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J. Virol. 79, 3787-3796] that chimeric virus 319.1v containing the E2 glycoprotein gene from Classical Swine Fever Virus (CSFV) vaccine strain CS with the genetic background of highly virulent CSFV strain Brescia (BICv) was markedly attenuated in pigs. To identify the amino acids mediating 319.1v attenuation a series of chimeric viruses containing CS E2 residues in the context of the Brescia strain were constructed. Chimera 357v, containing CS E2 residues 691 to 881 of CSFV polyprotein was virulent, while chimera 358v, containing CS E2 residues 882 to 1064, differing in thirteen amino acids from BICv, was attenuated in swine. Single or double substitutions of those amino acids in BICv E2 to CS E2 residues did not affect virulence. Groups of amino acids were then substituted in BICv E2 to CS E2 residues. Mutant 32v, with six substitutions between residues 975 and 1059, and mutant 33v, with six substitutions between 955 and 994, induced disease indistinguishable from BICv. Mutant 31v, with seven substitutions between residues 882 and 958, induced a delayed onset of lethal disease. Amino acids abrogating BICv virulence were then determined by progressively introducing six CS residues into 31v. Mutant 39v, containing nine residue substitutions, was virulent. Mutant 40v, containing ten residue substitutions, induced mild disease. Mutant 42v, containing twelve substitutions, and mutant 43v, with an amino acid composition identical to 358v, were attenuated in swine indicating that all substitutions were necessary for attenuation of the highly virulent strain Brescia. Importantly, 358v protected swine from challenge with virulent BICv at 3 and 28 days post-infection.