Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca²⁺ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P₁ position of peptide substrates and that this is most probably a result of hydrogen bonding from the P₁ residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P₂ and P₃ positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca²⁺ on the enzyme revealed the presence of two Ca²⁺ binding sites and a reversible structural modification of the enzyme upon Ca²⁺ binding. In addition, the concentration of Ca²⁺ used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P₂ were examined, suggesting that Ca²⁺ activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S₂ binding pocket. Collectively, these data give new insights into the substrate specificity and Ca²⁺ activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.