The mechanism of ISA23 · HCl interaction with model membrane vesicles (80–100 nm in diameter) was investigated using EPR in conjunction with SANS. For EPR, 16-DSE was dissolved in the vesicle membrane to measure its dynamics and polarity, whereas a spin-labeled (Tempo)-ISA 23 analogue was used to give a measure of the polymer flexibility. When ISA23 was added to the external vesicle surface, no interaction was found. This observation conflicts with the reported ability to lyse RBC, but is in agreement with recent studies that showed no effect on membrane permeability when a PAA was added to an incubation medium containing isolated lysosomal vesicles. The vesicle-mimetic models used here provide a new and useful tool for studying endosomolytic polymer/membrane interactions.