Metallocarboxypeptidases (MCPs) of the M32 family， while broadly distributed among prokaryotic organisms， have so far been only found in a few eukaryotes including trypanosomatids. Among these organisms are human and animal pathogens of medical relevance such as Trypanosoma brucei and Trypanosoma cruzi， the respective causative agents of sleeping sickness and Chagas disease. The M32 MCP orthologues found in these parasites share 72% protein sequence identity. They also present a cytosolic localization， a similar pattern of expression and a marked preference for Arg/Lys residues at P1'. To further explore MCPs substrate specificity beyond the S1' subsite， we employed four positional scanning synthetic combinatorial libraries (PS-SC) of fluorescence resonance energy transfer (FRET) peptides. Our results indicated that the T. brucei enzyme has a restricted selectivity for Phe in P1 position compared to T. cruzi MCP-1， which presented a wider range of substrate acceptance. The S2， S3 and S4 subsites， on the other hand， could accommodate a broad range of residues. On the basis of these results， we synthesized for each enzyme a series of FRET substrates which contained the most favourable residues in every position. In particular， for both MCPs acting on FRET pentapeptide substrates， catalytic efficiencies were ∼100 times higher compared with previously described chromogenic substrates. In fact， the fluorogenic peptide Abz-LLKFK(Dnp)-OH (Abz = ortho-aminobenzoic acid; Dnp = 2， 4-dinitrophenyl) described here can be used to monitor accurately TbMCP-1 activity in parasite cell-free extracts. These results provide valuable insights to develop selective substrates and inhibitors， to further understand the mechanisms and functions of M32 MCPs.