Rationale : Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined. Objective : Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation. Methods and Results : MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1 ^−/− mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1 ^−/− coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent. Conclusion : Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus).