Perfringolysin O (PFO) is an essential virulent factor of the bacterium Clostridium perfringens. It is secreted as a water soluble monomer and binds particularly to cholesterol-rich membranes. Research has showed that the cytolytic mechanism of PFO is initiated by binding of the domain 4 (D4) to the eucaryotic cell membrane and that the cholesterol recognition is mediated by four loops at the tip of the domain. A particular pair of amino acids, T490-L491, termed as cholesterol-recognition motif (CRM) in the literature, is essential for recognizing and binding to cholesterol within the membrane. Despite these findings, the exact mechanism of membrane binding and specific recognition of the membrane cholesterol on a molecular level is not fully understood. The aim of this thesis was to characterize the effects of amino acid substitutions in D4 loops of the protein PFO on binding to various membrane models. We have designed and expressed mutant variations of PFO and individual D4s in the E. coli bacterial expression system and successfully purified them with Ni-NTA affinity chromatography and size-exclusion cromatography. The effect of amino acid substitutions of PFO were characterized with hemolysis assays, whereby the activity of each mutant variation was compared to the activity of the wild type. We discovered that the mutant variations, containing more hydrophobic amino acids in the substituted locations in the CRM, have higher hemolytic activity. Binding assays to model membranes revealed that individual D4s with amino acid substitutions in the proposed CRM bind to multilamelar vesicles, as well as giant unilamelar vesicles with 50 mol. % cholesterol. We have discovered that despite substitutions of amino acids with the kind that have different biochemical properties, certain mutant variations of PFO can bind to cholesterol rich membranes.