In premature human neonates, immaturity of cerebral vessels can contribute to clinical problems such as germinal matrix hemorrhage and white matter damage. Afferent cerebral vessels in the brain of term babies express alkaline phosphatase (AP), an ectoenzyme located on the surface of endothelial cells. Using AP enzyme histochemistry we have examined the cerebrovasculature of premature live-born human neonates to determine when cerebral afferent vessels begin to express AP. Brains were collected at autopsy and processed for histological examination. AP-stained vessel density in the periventricular white matter was quantified using digital imaging and automated morphometry. Babies born prior to 28 wk gestation display few AP-positive vessels in the periventricular white matter, whereas, babies born after 28 wk gestation exhibit an AP-positive vascular pattern that resembles the adult pattern. In contrast, immunostaining for collagen revealed an extensive vascular network in both early and late gestation infants. Our measurements indicate that neonates born prior to 28 wk gestation are characterized by immature cerebral white matter afferent vessels and raise the possibility that the immaturity compromises vascular function.