Claudin-5 is a key component of tight junctions (TJs) in brain endothelial cells, which are essential for maintaining the blood-brain barrier (BBB). This study investigates the role of claudin-5 in BBB function by generating claudin-5-deficient mice. Despite normal vascular development and no signs of bleeding or edema, these mice exhibited size-selective loosening of the BBB, allowing small molecules (<800 D) to pass through but not larger ones. This suggests that claudin-5 is critical for maintaining the BBB's molecular sieve function. The BBB in these mice was confirmed to be compromised using tracer experiments and magnetic resonance imaging (MRI), showing increased permeability to small molecules. However, serum albumin and other larger molecules did not leak, indicating a size-selective barrier. The study also found that claudin-12, another claudin, remained at tight junctions in the absence of claudin-5, suggesting that claudin-12 may compensate for the loss of claudin-5 in maintaining TJ structure and function. These findings highlight the importance of claudin-5 in BBB integrity and suggest that targeting claudin-5 could be a potential strategy for developing new drug delivery methods for CNS disorders. The study also discusses the implications of BBB disruption, noting that while the BBB was compromised, no significant vasogenic edema was observed, indicating that the size-selective nature of the barrier may prevent excessive leakage. The research provides new insights into the molecular basis of BBB function and opens avenues for targeted drug delivery to the CNS.Claudin-5 is a key component of tight junctions (TJs) in brain endothelial cells, which are essential for maintaining the blood-brain barrier (BBB). This study investigates the role of claudin-5 in BBB function by generating claudin-5-deficient mice. Despite normal vascular development and no signs of bleeding or edema, these mice exhibited size-selective loosening of the BBB, allowing small molecules (<800 D) to pass through but not larger ones. This suggests that claudin-5 is critical for maintaining the BBB's molecular sieve function. The BBB in these mice was confirmed to be compromised using tracer experiments and magnetic resonance imaging (MRI), showing increased permeability to small molecules. However, serum albumin and other larger molecules did not leak, indicating a size-selective barrier. The study also found that claudin-12, another claudin, remained at tight junctions in the absence of claudin-5, suggesting that claudin-12 may compensate for the loss of claudin-5 in maintaining TJ structure and function. These findings highlight the importance of claudin-5 in BBB integrity and suggest that targeting claudin-5 could be a potential strategy for developing new drug delivery methods for CNS disorders. The study also discusses the implications of BBB disruption, noting that while the BBB was compromised, no significant vasogenic edema was observed, indicating that the size-selective nature of the barrier may prevent excessive leakage. The research provides new insights into the molecular basis of BBB function and opens avenues for targeted drug delivery to the CNS.