We hypothesized that modulation of the effective charge density of the endothelial surface layer (ESL) results in altered arterial barrier properties to transport of anionic solutes. Rat mesenteric small arteries (d ~190 µm) were isolated, cannulated, perfused and superfused with MOPS-buffered physiological saline solutions. MOPS-solutions were of normal ionic strength (MOPS, IS = 162 mM), low ionic strength (LO-MOPS, IS = 81 mM), or high ionic strength (HI-MOPS, IS = 323 mM), to modulate ESL charge density (respectively normal, high or low ESL charge). Osmolarity of MOPS, LO-MOPS and HI-MOPS was kept constant at 297 mosmol, using additional glucose when necessary. Perfusate solutions were supplemented with 1% bovine serum albumin. Arteries were cannulated with a double-barreled -pipet on the inlet side and a regular pipet on the outlet side. After infusion of fluorescein-isothiocyanate (FITC)-labeled dextran of 50 kD (FITC-50) and the endothelial membrane dye DiI, dynamics of arterial dye-filling were determined with confocal microscopy. ESL thickness, as determined from the initial exclusion zone for FITC-50 on the luminal endothelial surface, was 6.3 ± 1.4 µm for LO-MOPS, 2.7 ± 1.0 µm for MOPS, and 1.1 ± 1.3 µm for HI-MOPS. At low ionic strength, FITC-50 permeated into the ESL with a total ESL permeation time (ESL) of 26 min, and at normal IS with a ESL of 20 min. No apparent exclusion of FITC-50 from the ESL could be observed at high IS. In conclusion, we demonstrate that modulation of solvent ionic strength influences the thickness and barrier properties of the ESL.