Cell culture models have been widely used for screening of neurotoxicants and represent a viable alternative to direct in vivo experiments. We have developed a dynamic in vitro blood-brain barrier model designed to allow for extensive toxicological, pharmacological and physiological testing. Induction of blood-brain barrier properties in a tri-dimensional hollow fiber culturing apparatus was investigated by co-culturing a bovine aortic endothelial cell line (or rat brain endothelial cells) with rat brain astrocytes (or C6 rat glioma cells) under pulsatile flow conditions to mimic intraluminal blood flow. Cell growth was monitored over time by measuring glucose consumption and lactate production: these experiments confirmed that the hollow fiber cell culturing systems can maintain viable cells in culture for extended (> 1 month) periods of time. Cells were visually inspected after culturing and dissociation from the hollow fiber cartridge and identified as endothelial (by fluorescent Dil-Ac-LDL uptake) or glial (by GFAP immunoreactivity). Blood-brain barrier properties were tested by intraluminal injection of horse-radish peroxidase (HRP, mol. weight 44,000), glucose (m.w. 180) or potassium. Either procedure demonstrated that aortic cells co-cultured with astrocytes (or C6 cells) developed a selective barrier with an estimated electrical resistance of 2,900 omega/cm2. The electrophysiological and morphological properties of BAEC were also affected by the co-culturing process, suggesting that astrocytes induced CNS properties in these cells. These results demonstrate that the hollow fiber cell co-culturing system may be used as a dynamic model of the mammalian blood-brain barrier.