We examined the expression of tight junction (TJ) proteins in cerebral cortex, cerebellum, and spinal cord of fetuses after maternal treatment with single and multiple courses of dexamethasone. Ewes received either single courses of four 6-mg dexamethasone or placebo injections every 12-h for 48-h between 104-107 days, or the same treatment once a week between 76-78 and 104-107 days gestation. TJ protein expression was determined by Western immunoblot on tissue harvested at 105-108 days gestation. Blood-brain barrier permeability was previously quantified with the blood-to-brain transfer constant (K_i) with -aminoisobutyric acid (39). After a single course of dexamethasone, claudin-5 increased (P<0.05) in cerebral cortex, occludin and claudin-1 increased in cerebellum, and occludin increased in spinal cord. After multiple dexamethasone courses, occludin and zonula occludens (ZO)-1 increased in cerebral cortex, and occludin and claudin-1 in cerebellum. JAM-A and ZO-2 expressions did not change. Linear regression comparing K_i to TJ proteins showed inverse correlations with claudin-1 and claudin-5 in cerebral cortex after a single course, and ZO-2 in spinal cord after multiple courses, and direct correlations with ZO-1 in cerebellum and spinal cord after multiple courses. We conclude that maternal glucocorticoid treatment increases the expression of specific TJ proteins in vivo, patterns of TJ protein expression vary after exposure to single and multiple glucocorticoid courses, and decreases in blood-brain barrier permeability are associated with increases in claudin-1, claudin-5 and ZO-2 expression, and decreases in ZO-1 expression. In utero glucocorticoid exposure alters the molecular composition of the barrier and affects fetal blood-brain barrier function.