The increasing number of newly developed drugs demands for functional in vitro models of the blood-brain barrier to determine their brain uptake. Cultured cerebral capillary endothelial cells are considered to be such a model, however in serum containing media they exhibit low electrical resistances and high permeabilities compared to the in vivo situation. Here we report the establishment of a serum-free cell culture model. Withdrawal of serum already caused a twofold increase of transendothelial resistance (TER), which in presence of serum is about 100-150 omega.cm2. We tested several supplements and found that hydrocortisone is a potent stimulator for the formation of barrier properties. TERs up to 1000 omega.cm2 were measured in the presence of physiological relevant hydrocortisone concentrations. In correspondence to the TER increase hydrocortisone decreased cell monolayer permeability for sucrose down to 5 x 10(-7) cm/s, which is close to the in vivo value of 1.2 x 10(-7) cm/s and by a factor of five lower compared to cultures without hydrocortisone and in presence of serum.