To determine the differential effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight(.)day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.