We investigated the mechanism of hyperreactivity to collagen of the platelets from hypercholesterolemic rabbits, as compared with cholesterol-enriched platelets prepared in vitro by incubation of normal platelets with hypercholesterolemic plasma or cholesterol-lecithin dispersion. The platelets from rabbits fed high cholesterol diet for only 1 to 2 weeks, had an enhanced reactivity in aggregation and secretion, as well as an increase in cholesterol content and a decrease in membrane lipid fluidity. The cholesterol-enriched platelets prepared in vitro also had the same properties. In all these cholesterol-enriched platelets, two membrane functions, Ca2+-influx and arachidonate liberation from membrane phospholipids, were markedly enhanced in response to collagen, probably due to the influence of cholesterol upon physical properties of the membranes. These data suggest that in platelets from hypercholesterolemic rabbits, the decrease in membrane fluidity, accompanying the increase in membrane cholesterol content, brings about the enhancement of Ca2+-influx and liberation of arachidonate, thus resulting in hypersensitivity to collagen.