An increased sensitivity to epinephrine-induced aggregation has been observed both in platelets obtained from patients with type IIa hyperlipoproteinemia and in normal platelets following incubation with cholesterol-rich lecithin dispersions. We have reported previously that the membrane fraction of platelets is enriched with cholesterol relative to phospholipid under each of these conditions. To further explore the effect of cholesterol on platelet membranes, we have examined the fluidity (microviscosity) of whole platelets and platelet subcellular fractions using a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), under conditions in which the cholesterol-to-phospholipid mole ratio (C/PL) of platelets was varied by incubation with various cholesterol-lecithin sonicated dispersions. The C/PL of platelets directly influenced the rotational diffusion of DPH, as indicated by changes in fluorescence polarization. This was reflected in an increase in microviscosity at 37 degrees C (ETA37) from 2.84 P in normal platelets to 4.06 P in platelets with a 118% increase in C/PL. Conversely, platelets with a 43% decrease in C/PL had a 13% decrease in eta37. A strong correlation (r = 0.94) existed between C/PL and eta37 throughout this entire range. However, C/PL had no effect on the excited-state fluorescence lifetime of DPH. Both C/PL and eta37 were lower in isolated platelet membranes than in the platelet granule fraction. When platelets were incubated for 20 h with cholesterol-rich dispersions, there was an increase in C/PL and eta37 in both the membrane and granule fractions. However, this occurred more rapidly in membranes so that, at 5 h (a time when an increased sensitivity of whole platelets to epinephrine is evident), membrane C/PL had increased 55% and eta37 had increased 42%, whereas granule C/PL and eta37 had changed minimally. Cholesterol-rich platelets and subcellular fractions had a lower fusion (or flow) activation energy for viscosity (deltaE), reflecting a higher degree of order, and the converse was true in cholesterol-poor platelets. Moreover, a strong negative correlation existed between the percent change in deltaE and the percent change in eta37 induced either by cholesterol incorporation or depletion. These data demonstrate that cholesterol influences the fluidity and the degree of order within the hydrophobic core of platelet membranes. Changes induced in these physical properties by an excess of cholesterol relative to phospholipid may underlie the abnormal reception or transmission of the aggregation stimulus in cholesterol-rich platelets.