Transposon linker insertion mutagenesis of a full-length infectious clone of the highly pathogenic classical swine fever virus (CSFV) isolate Brescia (pBIC) was used to identify genetic determinants of CSFV virulence and host range. A virus mutant, RB-C22 (RB-C22v), possessing a 19-residue tag insertion at the carboxyl end of E1 was constructed. RB-C22v and the parental virus pBIC (pBICv) exhibited similar growth characteristics on primary porcine macrophage cell cultures although RB-C22v produced significantly smaller plaques on SK6 cell cultures. In vivo, RB-C22v was markedly attenuated in swine. In contrast with pBIC infection, where mortality was 100%, all RB-C22v-infected pigs survived infection remaining clinically normal. Additionally, chimeras of the Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Chimeras 138.8v and 337.14v, chimeras containing the E2 glycoprotein of CS and chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, were attenuated in swine. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. The combined results indicate a significant role for E1 glycoprotein and E2 glycoprotein in swine virulence.