Brain development occurs in a specialized environment maintained by a blood–brain barrier (BBB). An important structural element of the BBB is the endothelial tight junction (TJ). TJs are present during the embryonic period, but BBB impermeability accrues over an extended gestational interval. In studies of human premature infants, we used immunomicroscopy to determine if amounts of the TJ proteins ZO-1, claudin and occludin increase with gestational age in vessels of germinal matrix (GM) and cortex. By 24 weeks postconception (PC), TJ proteins were present in both GM and cortical vessels, but immunoreactivity in the GM of the youngest subjects was less than in older subjects. At 24 weeks PC, TJ protein immunoreactivity in GM vessels was less than in cortical vessels suggesting that TJ maturation progresses along a superficial to deep brain axis. This concept correlates with conclusions from previous analyses of the expression of brain endothelial cell alkaline phosphatase (AP) activity. AP appears in cortical vessels before appearing in deep white matter and GM vessels. Together, these data indicate that differentiation of some functional specializations is still in progress in GM vessels during the third trimester. This maturation could relate to the pathogenesis of germinal matrix hemorrhage–intraventricular hemorrhage.