The function of a 12-nt insertion 'CTTTTTTCTTTT' in the 3' untranslated region (3'UTR) of the HCLV strain, a vaccine strain derived from the Shimen strain of classical swine fever virus (CSFV), was examined in vitro. RNA synthesis increased when the 12-nt insertion was deleted from the 3'UTR of the HCLV strain. RNA synthesis also decreased when the 12-nt insertion was introduced into the 3'UTR of the CSFV Shimen, virulent strain. Therefore, the 12-nt insertion present in the 3'UTR of the HCLV strain may be a marker for avirulence. It was also found that the predicted secondary structure of the 3'UTR of the HCLV strain was more stable when the 12-nt insertion was deleted, and that the 3'UTR of Shimen strain was more unstable when the 12-nt insertion was introduced. Replacement of the 12-nt insertion 'CTTTTTTCTTTT' by another 12-nt fragment 'ATTATTATTTAT' in the 3'UTR of HCLV strain facilitated RNA synthesis and stabilized the predicted secondary structure, which was also observed in the 3'UTR of the Shimen strain.