The relationship of free arachidonic acid (AA) to cellular permeability, lipid peroxidation and physical state "fluidity" of the membrane was investigated in cultured endothelial cells (EC) dissociated from cerebral microvessels of rats. The results demonstrate that AA can induce a reversible alteration of endothelial permeability to trypan blue albumin (TBA). Exposure of EC to AA increases membrane "fluidity" as measured by fluorescence anisotropy using 1,6-diphenyl-1,3,5 hexatriene as a fluorescent probe. The AA modification of EC membrane "fluidity" is not associated with changes in EC permeability. Addition of AA and H2O2 to the incubation medium of EC leads to persistant alteration of EC permeability which can be prevented by catalase treatment. Both AA and H2O2 induce a greater formation of malondialdehyde, the product of lipid peroxidation, than AA alone. These findings strongly suggest that a release of AA either from the capillary or cellular membrane of the brain under a pathological condition may alone or through a peroxidative process alter the function of blood-brain barrier.