Endothelial cell contraction plays a pivotal role in the increased extravasation of fluid and macromolecules in vascular leakage. Previous studies have indicated that elevation of the adenosine 3’,5’-cyclic monophosphate (cAMP) concentration can improve the endothelial barrier function. In analogy with smooth muscle cell contraction, which is inhibited by both cAMP and guanosine 3’,5’-cyclic monophosphate (cGMP), we have compared the role of cAMP and cGMP in the regulation of the permeability of human endothelial cell monolayers. The cellular cGMP concentration was elevated 3- to 5-fold after addition of 10<sup>–7</sup> M atrial natriuretic peptide (ANP) or 10<sup>-4</sup> M sodium nitroprusside (SNP), both under basal and thrombin-stimulated conditions. After exposure to thrombin, cGMP generation by ANP or SNP or addition of 8-bromo-cGMP significantly suppressed the increase in permeability. Inhibition of nitric oxide production with 10<sup>–4</sup> M N<sup>G</sup>-nitro-L-arginine methyl ester increased the permeability of endothelial monolayers in the majority of the tested cultures, an effect that could be counteracted by addition of 8-bromo-cGMP or ANP. An increase of cAMP upon the addition of forskolin reduced the permeability in all endothelial cell strains under basal conditions and after exposure to thrombin. The forskolin- and 8-bromo-cGMP-mediated decreases in permeability were accompanied by increases in transendothehal electrical resistance. These in vitro data indicate that, in addition to cAMP, cGMP can act as a potent fine-regulator of endothelial permeability.