Fourth passage rat brain capillary endothelial cell cultures, which no longer possess the tight junctions characteristic of this highly specialized component of the blood-brain barrier, were used to study induction of zonulae occludentes in vitro. These cells, when grown in 50% rat brain astrocyte-conditioned medium and 50% alpha-MEM on an endothelial cell matrix-coated substrate (Cedarlane Labs, Hornby, Ont.), possessed numerous, elaborately complex, tight junctions which were identical to those displayed in vivo by intact brain capillary endothelium. Endothelial cells grown in 50% astrocyte-conditioned medium and 50% alpha-MEM on bare plastic or fibronectin-coated substrate, possessed no tight junctions. Results of this study clearly demonstrate the local control of tight junction biogenesis in brain capillary endothelial cells depends on: (1) an astrocyte-produced factor(s), and (2) a 'competent' (cell-produced) extracellular matrix.