The structural equivalent of the blood-brain barrier are the complex tight junctions (TJs) between endothelial cells of brain capillaries. In this study, we have quantitatively investigated by the freeze-fracture technique the modulation of the fine structure of TJs in blood-brain barrier endothelial cells during development of the rat cerebral cortex. The complexity of the TJ network as defined by fractal dimension, the integrity of TJ strands and the degree of TJ particle association to the protoplasmic leaflet of the membrane bilayer in percent of total TJ length were evaluated at embryonic days (E) 13, 15, 18, postnatal day (P) 1 and adult. We observed that the overall complexity of the TJ network and P-face association of TJ particles are significantly increased between E18 and P1. The increase in both of these TJ parameters in combination with the completed particle insertion starting from E18 is likely to reflect the process of transition to the mature state of the blood-brain barrier, which is characterized by high complexity of TJs and predominance of P-face association of TJ particles and correlated tightly with previous physiological measurements, e.g. transendothelial electrical resistance. Two populations of TJs differing in TJ particle density were distinguishable at E15 and E18, which indicates a non-linear asynchronous mechanism of TJ assembly. At E13, particle-free membrane specializations arranged in a TJ-like pattern strongly resembled TJ specific grooves and ridges. Similar results were obtained from cultures of brain endothelial cells in the presence of low calcium conditions, which suggests the involvement of the cadherin/catenin complex in TJ regulation. The particle-free 'TJ precursors' strongly indicate an established TJ associated cytoskeletal network before the TJ particles are present in their intra-junctional location.