The structural basis for the decline in non-specific permeability (tightening) of the blood-brain barrier (bbb) during development was investigated in fetal, newborn and weanling mice cerebral hemispheres. Permeability was assessed by measuring the peroxidase activity in the brain 4 h after i.p. injection of horseradish peroxidase--a commonly used vascular tracer that does not cross the intact bbb. Peroxidase activity in brain declined by about two-thirds between birth and weaning, despite increases in both circulating levels and vessel density. Morphometric analysis of the cerebral vessels showed that vesicular density was low (less than 4/micron 2 cytoplasm) at all ages examined , and therefore, is unlikely to be related to permeability changes. Interendothelial junctions changed in that the proportion of the junction composed of zonulae occludens increased, while junctional clefts decreased, and expanded junctional clefts virtually disappeared. We conclude, therefore, that junctional changes underlie the developmental tightening of the bbb. The observation that junctional changes also showed a strong inverse correlation with vessel density suggests that junctional leakiness, and therefore high non-specific permeability, may be a consequence of vessel proliferation in the developing brain.