Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood–brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-α, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-α treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-α and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-α was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1β. Concomitantly, TNF-α induced IL-1β mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1β mRNA synthesis and IL-1β protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-α and IL-1β between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.