The binding of cholesterol by pneumolysin, alveolysin, and streptolysin 0 has been demonstrated. The properties of the cytolysin-cholesterol interaction parallel those of cytolysin-erythrocyte interaction in that the reaction is rapid, temperature independent, decreased at elevated pH, and shows the same specificity with respect to other related sterols. However, oxidized or p-hydroxymercuribenzoate-treated toxin showed no decrease in cholesterol-binding activity, whereas the ability of cytolysin to bind to erythrocytes was modified by such treatment. One of the several properties common to that group of cytolytic agents known as the sulflydryl (or thiol)-activated toxins is sensitivity to inhibition by cholesterol and certain related sterols (see review by Bernheimer, 4). These agents act only on cells containing cholesterol (3), and it has been shown with one member ofthe group, cereolysin, that the interaction of toxin with liposomes depends on their cholesterol content (6). The work cited above supports the hypothesis that target cell membrane cholesterol is the receptor for the sulfhydryl-activated toxins. Although several reports of in vitro interactions between purified sulfhydryl-activated toxins and sterol dispersions h4ve appeared (7, 18, 21), there has been no direct demonstration of the binding of pure toxin with cholesterol. In the present communication we present evidence for such a binding and describe some properties of the system. MATERIALS AND METHODS Reagents. Cholesterol (cholest-5-en-3,8-ol) was obtained from Sigma Chemical Co. and from Steraloids, Ltd, [3H]cholesterol with a specific activity of 50 Ci/ mmol from New England Nuclear Corp. and the Commissariat de l'Energie Atomique (France), epicholesterol (cholest-5-en-3a-ol) and 7-dehydrocholesterol (cholsta-5,7-dien-3,8-ol) from Schwartz/Mann; bovine serum albumin from Pentex Biochemical; cytochrome c from Mann Research Laboratories; p-hydroxymercuribenzoate (pHMB) from Sigma Chemical Co.; oxidized dithiothreitol (DTT) from Calbiochem; and reduced DTT from Koch Light Laboratories. Purification of cytolysins. Pneumolysin was purified and titrated as described previously (11, 12