The structure and functions of the human erythrocyte are influenced by the composition and organization of the membrane lipids. The outer (exofacial) and inner (endofacial) leaflets of the erythrocyte membrane differ in lipid composition, and recent studies using a group of membrane-impermeant pyrene fluorophores have demonstrated that the lipid fluidity of the outer leaflet exceeds that of the inner. Using one of these probes, pyrene butyryl hydrazide linked to the tetrasaccharide stachyose (SPBH), we have compared the lipid fluidity of the outer and inner leaflets in normal human erythrocytes treated experimentally to alter membrane cholesterol content and in acanthocytes, erythrocytes of altered morphology found in individuals with the genetic disorder abetalipoproteinaemia. The results, reported here, demonstrate that hemileaflet fluidity can be altered selectively: acanthocytosis and experimental cholesterol enrichment decrease the lipid fluidity of the outer but not the inner hemileaflet.