Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion (EC GC-A KO mice) exhibit significant chronic hypervolemic hypertension (J Clin Invest 2005; 115: 1666). The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to FITC-labelled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nM) increased microvascular FITC-BSA extravasation in control but not in EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg/min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure (CVP) of control mice, but rapidly and significantly increased CVP of EC GC-A KO mice. In cultured lung endothelial cells ANP provoked cGMP-dependent protein kinase I (PKG I) - mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by PKG I-dependent phosphorylation of VASP. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.