Atrial natriuretic peptide (ANP) is a peptide hormone released from the cardiac atria during hypervolemia. Though named for its well-known renal effect, ANP has been demonstrated to acutely increase vascular permeability in vivo. Experimentally, this phenomenon was associated with a marked shedding of the endothelial glycocalyx, at least for supraphysiological intravascular concentrations. This study investigates the impact and mechanism of action of physiological doses of ANP and related peptides on the vascular barrier. In isolated guinea pig hearts, prepared and perfused in a modified Langendorff mode with and without the intravascular presence of the colloid hydroxyethyl starch (HES), we measured functional changes in vascular permeability and glycocalyx shedding related to intracoronary infusion of physiological concentrations of A-, B- and C-type natriuretic peptide (ANP, BNP and CNP). Significant coronary venous washout of glycocalyx constituents (syndecan-1 and heparan sulfate) was observed. As tested for ANP, this effect was positively related to the intracoronary concentration. Intravascular shedding of the glycocalyx was morphologically confirmed by electron microscopy. Also, functional vascular barrier competence decreased, as indicated by significant increases in transudate formation and HES extravasation. Ortho-phenanthroline, a non-specific inhibitor of matrix metalloproteases, was able to reduce ANP-induced glycocalyx shedding. These findings suggest participation of natriuretic peptides in pathophysiological processes like heart failure, inflammation or sepsis. Inhibition of metalloproteases might serve as a basis for future therapeutical options.