Metacaspases are cysteine-dependent enzymes that belong to the same peptidase family as caspases, which are essential regulatory proteins involved in metazoan apoptosis, the programmed cell death that occurs in animals. Metacaspases share many characteristics with caspases, and thus are believed to be involved in the pathways of programmed cell death in fungi, protozoa, and plants. However, even though metacaspases play a similar role in apoptosis, they likely perform it through different means. Unlike caspases which show aspartate specificity, metacaspases exhibit a preference to cleave after arginine or lysine amino acid residues. We study the metacaspases found in the fungus, Schizophyllum commune, and have identified and purified five Type 1 S. commune metacaspases (ScMC1-5). Preliminary studies carried out with a limited number of substrates revealed that ScMC1-5 exhibit arginine/lysine specificity and do not cleave after aspartate but provided limited information about the amino acid preference at different binding pocket positions. To continue the investigation of ScMC1-5 substrate specificity, we performed in-vitro fluorescent activity assays to evaluate whether the metacaspases show preference towards specific amino acid residues in the peptide substrates. Specifically, we examined how amino acid size, charge and polarity affect the relative activity of the enzymes. ScMC1-5 demonstrate a preference to cleave more readily after arginine than lysine. However, the detailed substrate specificity profiles vary among these metacaspases. For example, we observed ScMC1 and ScMC4 recognize a wide variety of amino acid residues. Peptides with small non-polar amino acids like glycine, proline, valine, and alanine showed good relative activities, but the highest activity was toward a substrate with the small polar threonine. On the other hand, ScMC2,3, and 5 did not recognize most of those amino acid residues and the enzymes were inactive toward those substrates. They only showed some relative activity toward the large non-polar amino acid phenylalanine, the polar threonine, and had the highest activity toward the small non-polar glycine. Studying these small differences in substrate specificity can be relevant for determining the types of proteins that metacaspases cleave in vivo and for understanding their individual, physiological functions.