Metacaspases are caspase-like proteases found in fungi, plants, and bacteria. Structural and functional studies indicate that metacaspases cleave the peptide backbone in their natural substrates to aid in initiating programmed cell death (PCD) pathways. We are studying five metacaspases from the fungus Schizophyllum commune to better characterize their activation and efficiency. Metacaspase I from S. commune (ScMC1) is completely inactive without calcium ions, suggesting that calcium may allosterically activate the enzyme. Calcium binding sites are often formed by negative aspartic acid residues that can bind to the calcium cation. To identify residues involved with calcium binding we aligned the sequence of ScMC1 with previously characterized metacaspases from other organisms. Our analysis revealed six well conserved aspartic acid residues. Research on other metacaspases has suggested that these residues form two distinct calcium binding sites and that both of these sites may be required for enzyme activity. The high-affinity binding site is composed of four residues and binds tightly to calcium. In contrast, the two-residue low-affinity site does not bind calcium as well and therefore is not fully occupied until higher calcium concentrations. Each residue implicated in calcium binding was individually mutated to alanine, a residue that cannot interact with calcium, via site-directed mutagenesis. To characterize our enzymes we performed in vitro activity assays that quantify activity on large and small substrates over different calcium concentrations. We compared the activity of the mutants to that of the wild type enzyme. Regardless of the substrate used, ScMC1 is inactive without calcium. The calcium-binding mutants had very low activity over all substrates and calcium concentrations. These assays demonstrate that mutations to the high-affinity binding site inactivate the enzyme. Preliminary results with the low-affinity binding site mutants imply that calcium binding at both sites is required for metacaspase activity. The highly conserved and dramatic nature of calcium dependency suggests that calcium plays a significant role in ScMC1 activation in vivo. A better understanding of calcium-dependent metacaspase activation may illuminate cell death signaling pathways in medically and agriculturally relevant organisms.