The foot-and-mouth disease virus Leader proteinase (L(pro)) frees itself from the growing viral polyprotein by self-processing between its own C-terminus and the N-terminus of the subsequent protein VP4. The ArgLysLeuLys*GlyAlaGlyGln sequence is recognized. The proteinase subsequently cleaves the two isoforms of host cell protein eukaryotic initiation factor (eIF) 4G at the AlaAsnLeuGly*ArgThrThrLeu (eIF4GI) and LeuAsnValGly*SerArgArgSer (eIF4GII) sequences. The enzyme does not, however, recognize the sequence on eIF4GII (AlaAspPheGly*ArgGlnThrPro) which is analogous to that recognized on eIF4GI. To investigate the basis for this specificity, we used site-directed mutagenesis to show that the presence of Phe at the P2 position or Asp at the P3 position severely compromises self-processing. Furthermore, these substitutions also give rise to the production of aberrant cleavage products. As Leu is the preferred amino acid at P2, the specificity of L(pro) is reminiscent of that of cathepsin K. This cellular proteinase can also process collagen through its ability to accept proline at the P2 position. Investigation of the L(pro) substrate specificity showed, however, that in contrast to cathepsin K, L(pro) cannot accept Pro at P2 and does not cleave collagen. Subtle variations in the arrangement of the S2 binding pockets on the enzymes are responsible for these differences in specificity.