1  Department of Medical Physics, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; and 2  Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22906-0011 We previously reported that a 0.4- to 0.5-µm-thick endothelial surface layer confines Dextran 70 (70 kDa) to the central core of hamster cremaster muscle capillaries. In the present study we used a variety of plasma tracers to probe the barrier properties of the endothelial surface layer using combined fluorescence and brightfield intravital microscopy. No permeation of the endothelial surface layer was observed for either neutral or anionic dextrans 70 kDa, but a neutral Dextran 40 (40 kDa) and neutral free dye (rhodamine, 0.4 kDa) equilibrated with the endothelial surface layer within 1 min. In contrast, small anionic tracers of similar size (0.4-40 kDa) permeated the endothelial surface layer relatively slowly with half-times ( 50 ) between 11 and 60 min, depending on tracer size. Furthermore, two plasma proteins, fibrinogen (340   kDa) and albumin (67 kDa), moved slowly into the endothelial surface layer at the same rates, despite greatly differing sizes ( 50    40 min). Dextran 70, which did not enter the glycocalyx over the course of these experiments, entered at the same rate as free albumin when it was conjugated to albumin. These findings demonstrate that for anionic molecules size and charge have a profound effect on the penetration rate into the glycocalyx. The equal rates of penetration of the glycocalyx demonstrated by the different protein molecules suggests that multiple factors may influence the penetration of the barrier, including molecular size, charge, and structure. intravital microscopy; capillaries; endothelium; glycocalyx; solute barrier