Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5 degrees C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins.