Platelets from diabetic subjects and animals are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function and previously we observed reduced membrane fluidity in platelets from diabetic patients associated with hypersensitivity to thrombin. We previously reported that decreased fluidity of isolated platelet membranes from diabetic patients is associated with increased glycation of platelet membrane proteins, but not with any change in the cholesterol to phospholipid molar ratio. We have now examined in vitro whether incubation of platelet membranes in a high glucose medium causes sufficient glycation to reduce membrane fluidity. Incubation of platelet membranes from control subjects in a high glucose (16.1 mM) medium for 10 days at 37° C led to an increase in the extent of glycation of membrane proteins and a decrease in membrane fluidity (indicated by an increase in steady state fluorescence polarization); most of the changes occurred within the first 3 days of incubation. Incubation of platelet membranes with 5.4 mM glucose had less effect. In contrast, incubation of platelet membranes with the same concentrations of 1–0-methylglucose did not cause a change in either the extent of glycation of proteins or membrane fluidity. We also determined if acetylation by aspirin or acetyl chloride of the sites available for glycation on platelet membrane proteins leads to a similar reduction in membrane fluidity. Pretreatment of platelet membranes with aspirin or acetyl chloride diminished the extent of glycation that occurred when platelet membranes were subsequently incubated with glucose, but membrane fluidity was reduced even in the absence of glucose; subsequent incubation with glucose caused no further reduction in membrane fluidity. Similar results were obtained when red blood cells were incubated with high concentrations of glucose or methyl glucose either with or without pretreatment with aspirin or acetyl chloride. Further experiments using platelet membranes showed that the reduction in membrane fluidity due to aspirin was independent of its acetylating effect on platelet cyclo-oxygenase. Ingestion of aspirin also caused a reduction in membrane fluidity of platelets. Therefore, glycation of platelet membrane proteins reduces membrane fluidity, but the effect results from occupation of the sites available for glycation and not the presence of glucose moieties per se at these sites. Acetylation of platelet membrane proteins either in vitro or in vivo also reduces membrane fluidity; this effect is not associated with platelet hypersensitivity to thrombin.