Distinct aggregates of small intramembranous particles and assemblies characterize the P-face of freeze-fractured astrocytic membranes. To test the lability of the assemblies, astrocytes were treated in vitro with different chemical agents and in vivo by cold injury. The assemblies appeared either to contain or be associated with protein because exposure to medium containing cycloheximide, an inhibitor of protein synthesis, led to a sharp decrease in assemblies, down to 1% of the control levels within three hours. To ascertain whether the assemblies were tethered to the cytoskeleton, the cells were treated in vitro with disruptors of microtubules (colchicine) or microfilaments (cytochalasins); the assemblies became consistently rearranged. Protein denaturants, urea and guanidine HCl, brought about a selective aggregation of assembly with assembly. The lectin, concanavalin A, did not alter the distribution of the assemblies within the plane of the membrane fracture. Surface replicas of in vitro, non-fractured, astrocytes revealed surface particles which did not resemble assemblies. In vivo, the plasma membranes of astrocytes were altered within minutes of cold injury to the brain surface. In the centre of the lesions, damaged astrocytes had assemblies that were clumped like those of in vitro astrocytes exposed to denaturants. In the periphery of the lesions, however, the assemblies did not aggregate but increased in number. These results provide indirect evidence that assemblies may consist of protein, that the recognizable particle constituent of the assembly is confined to the interior of the membrane and is not present on the uncleaved cell surface, and that assemblies are connected with the cytoskeleton. Therefore, certain changes in the environment of the astrocyte caused by injury in vivo or addition of chemical agents in vitro alter the distribution of assemblies in the astrocytic plasma membrane either by a direct effect on the assemblies or indirectly by an alteration of the cytoplasmic proteins.