Thimet oligopeptidase (TOP; EC3.4.24.15) and neurolysin (EC3.4.24.16) are closely related metalooligopeptidases. These oligopeptidases could be involved in the metabolism of biological active peptides and recent works have shown that TOP participates in the selection of citosolic peptides in MHC class I antigen presentation route. The crystallographic structure of these peptidases associated with detailed substrate specificity studies indicated that the substrate binding regions of both oligopeptidases are composed by potentially flexible structures. In order to better understand this specificity and dissect the importance of specific amino acid residues in this flexibility and/or substrate binding and catalysis we thus produced mutant enzymes applying site-direct mutagenesis based on the crystallographic structures and in previous results. In this work we report results of kinetic studies using fluorogenic substrates and mutants TOP Y605F, TOP Y605A, neurolysin Y606F and neurolysin Y606A in comparison with the wild-type TOP and neurolysin respectively. Modeling studies indicates that Tyr605 (TOP) or Tyr606 (neurolysin) are located in the substrate binding sites of the respective peptidases in position to interact with the P₁ residue of their substrates. The results obtained with a series of substrates containing different amino acid residues at the P₁ position based in a previously developed substrate series Abz-GFSXFRQ-EDDnp are presented (cleavage at X-F bond; X = natural amino acid residues). Significant differences were observed comparing wild-type and mutant enzymes demonstrating the central role of the Tyr605 or Tyr606 in substrate recognition by TOP and neurolysin respectively. In general, increases in the K_M were observed for both TOP Y605A and neurolysin Y606A, but also significant differences in the catalytic constants were observed. It is noteworthy that TOP mutant Y605A presented differences in the cleavage site in comparison with wild-type enzyme.