The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3Cpro and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3Cpro while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3Cpro independently.