Glycosylphosphatidylinositol (GPI) anchored proteins are a structurally and functionally diverse group of eukaryotic proteins that localize to small ordered domains (lipid rafts) at the membrane surface. Bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves the GPI anchors of mammalian proteins and releases them from the membrane. The interfacial binding surface (IBS) of the PI-PLC interacts with the membrane via hydrophobic and charged residues. For rapid purification of PI-PLC, C-terminal His-tagged versions of wild type and mutant PI-PLCs (K44A, W47A, W242A, and W47A/W242A) were constructed. Circular dichroism (CD) spectroscopy showed that introduction of His-tag and site-specific mutations did not distort the protein secondary structure. Fluorescence studies showed the importance of two Trp residues in the interaction of PI-PLC with lipid bilayers. All four mutant PI-PLCs showed a decreased ability to cleave GPI-anchored protein from reconstituted proteoliposomes. These results suggest the importance of both electrostatic and hydrophobic interactions in PI-PLC membrane binding and catalysis.