Initiation of protein synthesis on the foot-and-mouth disease virus RNA occurs at two sites, thus, two forms of the leader protein, termed Lab and Lb, are produced. Plasmids have been constructed which encode these proteins either together or individually. Plasmids encoding the Lab protein alone express a modified form of this protein in which the second methionine residue, which corresponds to the first amino acid of Lb, is changed to an alternative residue. Four different mutant forms of the Lab sequence were made. Each of the plasmids was introduced into a mammalian cell transient expression system which allowed the determination of the known activities of the L proteins. It was shown that the Lb protein and each of the modified Lab proteins were capable of cleaving the L/P1 junction in trans. Furthermore, each of these proteins induced the cleavage of the p220 component of the cap-binding complex (eIF-4F) producing inhibition of cap-dependent translation. These results indicate that the two species of L have the same functions.