The Leader protein of foot-and-mouth disease virus (FMDV) is the first component of the virus polyprotein. It is synthesized in two forms, Lab and Lb, both of which display the ability to cleave the L/P1 junction in trans and to induce the cleavage of the cap-binding complex component eIF-4G (p220). The L protease has weak homology to the family of cysteine proteases, which have a catalytic dyad composed of a cysteine and a histidine. Mutations have been introduced into FMDV cDNA to modify each of the four cysteine residues and the three conserved histidine residues within the Lb species. The activities of the mutant L proteins have been determined. Modification of a single cysteine residue (residue 51) or of a single histidine residue (residue 148) abolished the abilities of L to cleave the L/P1 junction and to inhibit cap-dependent protein synthesis. In contrast, modification of each of the other cysteine residues and other conserved histidine residues had no apparent effect on these activities.