To explore the role in viral pathogenesis of the region located between the two functional AUG (inter-AUG) in foot-and-mouth disease virus (FMDV), we derived viruses containing transposon (tn) inserts from a mutagenized cDNA infectious clone of FMDV (pA24-WT). Mutant viruses containing an in-frame 57-nt transposon insertion grew at a slower rate and had a smaller plaque size phenotype than the parental virus (A24-WT). A mutant virus containing a 51-nt deletion in inter-AUG had a similar phenotype in cell culture to that of A24-WT. When tested by aerosol inoculation in cattle (3 animals per virus), the deletion mutant was fully virulent as was A24-WT. Mutant viruses containing insertions in inter-AUG did not cause clinical disease or viremia. However, viruses that partially or totally removed the tn insertion during animal infection reverted to virulence in 2 inoculated steers. Therefore, this study identified inter-AUG as an FMDV viral virulence determinant in cattle infected by aerosol route.