The toxin perfringolysin O (PFO) is an important virulence factor of the Gram-positive bacterium Clostridium perfringens. It belongs to the family of cholesterol-dependent cytolysins, which form large pores in target cell membranes. PFO binds to cholesterol in the membrane by using four loops located in domain 4 (D4). The amino acid motif T490 L491, in loop L1, plays an important role in cholesterol binding. The exact mechanism of membrane cholesterol recognition at the molecular level is not yet completely understood. The aim of this thesis was to characterize the effects of amino acid substitutions in D4 loops on the activity of PFO mutants. PFO mutants were produced in the bacterial expression system Escherichia coli and were successfully purified with Ni-NTA affinity and size-exclusion chromatography. We demonstrated that the amino acid sequences and molecular masses of the PFO mutants were, with the exception of one mutant, as expected. Surprisingly, we observed binding of PFO mutants with substitutions in the pair T490-L491 to multilamellar vesicles with 50 mol % cholesterol. On the other hand, the tested PFO mutants did not bind to vesicles with 50 mol % cholesteryl acetate. Furthermore, we determined the effectiveness of the PFO mutants by measuring hemolytic activity. The amino acid substitutions considerably changed the PFO mutants activity. Using surface plasmon resonance (SPR), we found that the A401W substitution increased PFO affinity for cholesterol, whereas amino acid substitutions in the cholesterol recognition motif lowered the affinity for cholesterol. In conclusion, the chosen PFO mutants can bind to cholesterol containing membranes, despite the substitutions in the proposed cholesterol binding motif.