The goal of the present study was to determine the role of basal synthesis/release of nitric oxide on the basal permeability characteristics of the blood–brain barrier to various sized molecules in vivo. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood–brain barrier was quantitated by calculating the clearance of fluorescent-labeled albumin (mol.wt.=69,000 Da; FITC–albumin), fluorescent-labeled dextran (mol.wt.=10,000 Da; FITC–dextran-10K) or sodium fluorescein (mol.wt.=376; NaFl) in the absence and presence of an inhibitor of nitric oxide synthase ( N G-monomethyl- l-arginine; l-NMMA; 10 and 100 μM). During superfusion with vehicle, clearance of FITC–albumin, FITC–dextran-10K and NaFl from pial vessels and diameter of pial arterioles remained constant. To determine whether basal synthesis/release of nitric oxide affected basal permeability of the blood–brain barrier, we examined the effects of l-NMMA (10 and 100 μM). In addition, we examined the adherence of leukocytes to cerebral venular endothelium using rhodamine 6G. Although topical application of l-NMMA produced constriction of pial arterioles, l-NMMA did not alter the permeability characteristics of the blood–brain barrier to FITC–albumin, FITC–dextran-10K or NaFl. Further, the adherence of leukocytes to the endothelium appeared to be similar while suffusing with vehicle and l-NMMA (100 μM). Thus, the findings of the present study suggest that while basal synthesis/release of nitric oxide may play an important role in regulation of basal tone of cerebral blood vessels, it does not appear that basal synthesis/release of nitric oxide plays an important role in maintaining the integrity of the blood–brain barrier to large or small molecules.