The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood-brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para- or transcellular permeabilities (Pe) of inulin (0.74 × 10−3 cm/min), sucrose (1.60 × 10−3 cm/min), lucifer yellow (1.33 × 10−3 cm/min), morphine (5.36 × 10−3 cm/min), propranolol (4.49 × 10−3 cm/min) and midazolam (5.13 × 10−3 cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1–5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1-mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p-glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood-brain barrier.