Technical Abstract: Foot-and-mouth disease virus (FMDV) is an extremely infectious and antigenically diverse picornavirus that causes high incidences of morbidity within naive livestock populations. Several reports indicate that FMDV O1 subtype strains undergo antigenic variation under diverse growth conditions. Of particular interest is the amino acid variation observed at position 56 within the structural protein VP3. Previous studies suggest that selective pressures influence whether arginine or histidine is present at this position, influencing in vitro plaque morphology and in vivo pathogenesis in cattle. Using reverse genetics techniques, we have constructed FMDV type O1 Campos variants differing only at VP3 position 56, possessing either an histidine (H) or arginine (R) (O1Ca-VP3-56H and O1 Ca-VP3-56R, respectively), and characterized their in vitro phenotype and virulence in the natural host. Viruses O1Ca-VP3-56H and O1Ca-VP3-56R had similar growth kinetics in BHK-21 and LF-BK cells and slight differences in plaque size in BHK, LF-BK, IBRS-2 and LK cells. In contrast, the viruses displayed significantly different pathogenicities in cattle and swine. The O1Ca-VP3-56H viruses induced typical clinical signs of FMD, whereas O1Ca-VP3-56R viruses were nonpathogenic unless they reverted in vivo to either H or Cysteine (C). These results indicate that the presence of R at position 56 of the FMDV VP3 protein plays a role in virus attenuation in the natural host.