Cholesterol is an essential component of mammalian cell membranes and it is important to regulate the structure and function of lipid bilayers. Changes in cholesterol levels are involved in many physiological and pathological events such as the formation of arterial plaques, viral entry into cells, sperm capacitation, and receptor organization. Determination of cholesterol trafficking and distribution is essential for understanding how cells regulate cholesterol. A key factor in the regulation of cholesterol is cholesterol accessibility. Through it interactions in the membrane, cholesterol is sequestered below the surface of the membrane. Based on the composition of the membrane, a certain amount of cholesterol can be solubilized through interaction with the membrane. The remaining cholesterol is more solvent exposed and therefor accessible to interact with molecules outside of the membrane. This accessible cholesterol is thought to regulate cholesterol homeostasis within the cell. A cholesterol probe capable of distinguishing changes in cholesterol accessibility within membranes would facilitate investigations in this area. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target cell membrane for binding. The specificity of PFO for high levels of accessible cholesterol makes this toxin a potential tool for the detection of cholesterol distribution and trafficking. In an effort to adapt PFO into a molecular probe capable of sensing changes in membrane cholesterol accessibility, I have taken a non-lytic derivative of PFO and introduced several point mutations in the membrane-interacting domain 4 loops. These mutations altered the threshold of cholesterol concentration required in the membrane to trigger binding. The cholesterol-dependent binding of each PFO derivative was tested on model membranes containing different percentages of cholesterol. Three PFO derivatives were selected to test their binding to macrophage plasma membranes. These three derivatives showed differential binding to cell membranes treated with β-methyl-cyclodextrin/cholesterol mixtures. These data showed that the produced PFO derivatives differentially bind to model and biological membranes containing different cholesterol accessibility.