The effect of the chemical modifications induced by aspirin (acetylsalicylic acid), acetyl chloride or salicylate on platelet membranes and erythrocyte ghosts has been investigated by means of fluorescence quenching and ESR spectroscopy in relation to our earlier findings of acetylation-induced reduction of platelet and erythrocyte membrane lipid fluidity. Only aspirin was found to induce disorders in the lipid-protein matrix and membrane protein conformation. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased after aspirin action in both platelet and erythrocyte membranes. This resulted in a significant increase in the maximum energy transfer efficiency. The decrease in the ratio of the amplitudes of low-field peaks of weakly to strongly immobilized fractions of maleimide spin label (4-maleimido-2,2,6-6-tetramethylpiperidine-1-oxyl) and the rise in the relative rotational correlation time of iodoacetamide spin label [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl] indicate that aspirin effectively immobilizes membrane proteins in the plane of the lipid bilayer, whereas neither acetyl chloride or salicylate gave rise to detectable effects. We conclude that aspirin-induced alterations in membrane protein structure induce a reorganization of lipid assembly as well as rearrangements in the membrane protein pattern, and consequently alterations in lipid-protein interactions. Thus, the interaction of aspirin with platelet and erythrocyte membranes may induce local conformational changes in membranes, which are discussed in connection with impairment of platelet function. A new mode of protein chemical modification by aspirin is suggested which involves the generation of reactive salicylic residue during the fast degradation of aspirin under physiological conditions.