We have evaluated a small water-soluble molecule, biotin ethylenediamine (BED, 286 Da), as a permeability tracer across the blood-brain barrier. This molecule was found to have suitable characteristics in that it is stable in plasma, has low plasma protein binding, and appears to behave in a similar manner across brain barriers as established by permeability markers such as sucrose. BED, together with a 3000-Da biotin-dextran (BDA3000), was used to investigate the effectiveness of tight junctions in cortical vessels during development and adulthood of a marsupial opossum (Monodelphis domestica). Marsupial species are born at an early stage of brain development when cortical vessels are just beginning to appear. The tracers were administered systemically to opossums at various ages and localized in brains with light and electron microscopy. In adults, the tight junctions restricted the movement of both tracers. In neonates, as soon as vessels grow into the neocortex, their tight junctions are functionally restrictive, a finding supported by the presence of claudin-5 in endothelial cells. However, both tracers are also found within brain extracellular space soon after intraperitoneal administration. The main route of entry for the tracers into immature neocortex appears to be via the cerebrospinal fluid over the outer (subarachnoid) and inner (ventricular) surfaces of the brain. These experiments demonstrate that the previously described higher permeability of barriers to small molecules in the developing brain does not seem to be due to leakiness of cerebral endothelial tight junctions, but to a route of entry probably via the choroid plexuses and cerebrospinal fluid.