Vesicle leakage experiments were carried out to establish how cholesterol content regulates membrane permeability as induced by surfactant exposure. Vesicles containing up to 50% cholesterol were examined. Four different surfactants were chosen as membrane perturbants, including nonoxynol-9 which is commonly used in spermicidal formulations. As part of this study, we establish that the extrusion procedure commonly used to fabricate unilamellar vesicles does not unintentionally alter the desired composition of these model membrane systems. The kinetics of the leakage process is well characterized by a single exponential rate of release, similar to the form seen in the absence of membrane cholesterol. Our leakage experiments show that membranes become more resistant toward surfactant attack, in direct proportion with cholesterol content. This rise in resistance is surfactant specific. Above 30%, all membranes show positive deviation from the linear increase in resistance with increasing cholesterol content. Two other sterols, dihydrocholesterol and coprostanol, were also found to increase membrane resistance and behaved similarly despite a key difference in molecular structure. A peculiar leakage response was observed when membranes were exposed to the surfactant sodium dodecyl sulfate (SDS) above its critical micelle concentration. Our findings support the hypothesis that SDS micelles solubilize phospholipid molecules, creating a membrane with higher cholesterol content that is extremely resistant to perturbation.