In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood-Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope=0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1beta) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.