The aggregation of human platelets by bovine Factor VIII (Platelet Agglutinating Factor-PAF) is inhibited by exposure of the cells to ADP or Chymotrypsin. We have investigated the mechanism of these effects using washed platelets. The washing procedure was modified from the method of Mustard et al. (Brit. J. Haematol. 22:193, 1972), omitting heparin and using a protein-free Tyrode’s solution for the final resuspension. The washed platelets were stable and responded to ADP (0.1-1 μM) with a shape change and, if fibrinogen was added, with aggregation. Bovine Factor VIII was purified to >90% homogeneity and was labeled with 125I (approx. 1 atom/subunit) by the IodoGen procedure, with no loss of activity. Aggregation was measured in the aggregometer in the presence of 7 mM EDTA. Binding was measured after incubation of labeled Factor VIII with washed platelets in the presence of 7 mM EDTA for 5 min at 37° without stirring.Treatment of washed platelets with Chymotrypsin progressively destroyed their ability to bind Factor VIII and to be agglutinated by it. Responsiveness to Factor VIII disappeared before any alteration was detected in the ability of platelets to undergo ADP-induced shape change. Treatment of platelets with ADP, however, inhibited agglutination induced by Factor VIII without affecting the binding of Factor VIII to the platelets. Agglutination by wheat germ agglutinin or phytohemagglutinin was not inhibited by ADP treatment. We conclude that Chymotrypsin probably inhibits Factor VIII- induced agglutination by destroying the platelet binding site for Factor VIII, but that ADP must act at a point distal to Factor VIII binding. Agglutination of metabolically intact platelets by Factor VIII may not be a simple process, because ADP can specifically inhibit it without affecting Factor VIII binding or aggregation of the platelets by lectins.