The endothelial cell-cell junctions of the blood-brain barrier (BBB) play a pivotal role in the barrier's function. Altered cell-cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell-cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a "global" and "local" scale.We cultured HBMECs on collagen I, fibronectin, collagen IV, laminin, fibronectin/collagen IV/laminin, or hyaluronic acid/gelatin for 2, 4, and 7 days with varying cAMP treatment schedules. Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk ("global") barrier properties, and a "local" permeability assay was used to correlate junction presentation proximal to permeable monolayer regions.Substrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach similar ZO-1 and VE-cadherin coverage observed with shorter culture, though longer cultures were required for claudin-5 presentation. Prolonged cAMP treatment (6 days) disrupted junction integrity for all three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the number of discontinuous and no junction regions with barrier penetration.These results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction phenotype and supported previous results that continuous junctions are indicative of a more mature/stable endothelial barrier. Understanding what conditions influence junction presentations, and how they, in turn, affect barrier integrity, could lead to the development of therapeutics for diseases associated with BBB dysfunction.