To ascertain whether there is a mutual influence on the structure of their cell membranes, brain endothelial cells and their closest neighbor, astrocytes, were grown alone or together in vitro and freeze-fractured. When cultured separately, the brain endothelial cells had a low frequency of short, fragmented tight junctions. Many gap junctions, which are absent from mature brain capillaries in vivo, intercalated among the tight junctional strands, or were separate from them. The separately cultured astrocytes had low concentrations of randomly distributed assemblies (1-30/micron2) in their membranes. When the two cell types were co-cultured, the endothelial tight junctions were greatly enhanced in frequency, length, width and complexity, and the gap junctional area enclosed by the tight junctional strands were markedly reduced. Thus, the in vitro endothelial junctional complex resembled their in vivo counterpart, the tight junctions of brain capillaries, when co-cultured with astrocytes. Reciprocally, brain endothelial cells induced the astrocytic membrane assemblies to increase in concentrations by approximately 5 fold, and sometimes to form aggregates with very high concentrations (400/micron 2) which approached the concentration of the perivascular astrocytic membranes in vivo. Substituting astrocytes with fibroblasts or smooth muscle cells in co-cultures did not enhance the tight junctions in the brain endothelium. On the other hand, substituting brain endothelium with endothelium from pulmonary artery or aorta in co-cultures did not increase the concentration or induce aggregation of the assemblies in the astrocytes. Thus, the two close neighbors in vivo, brain endothelium and astrocytes, interact specifically in vitro to induce development of membrane specializations which resemble those at the site of the blood-brain barrier.