We have developed a quantitative method to study the interaction of radiolabeled clathrin triskelions with membranes from brain coated vesicles. Clathrin triskelions do not bind to coated vesicles unless these are stripped of their clathrin coat. The binding of triskelions is of high affinity (KD ≅ 2 × 10-9 M), is saturable and depends on vesicle concentration. Triskelions bind to protein-sensitive structures of the stripped vesicles. Polypeptides of about 110,000 daltons appear to be involved in the binding structure. The association of triskelions to stripped vesicles was confirmed by electron microscopy.