Production of urine is initiated by fluid and solute flux across the glomerular filtration barrier. Recent ultrastructural studies have shown that under extreme conditions of no filtration, or very high filtration, a restriction to flow is predicted in a space underneath the podocyte cell body or its processes, the sub-podocyte space (SPS). The SPS covered up to two thirds of the glomerular filtration barrier (GFB) surface. The magnitude of this restriction to flow suggested that it might be unlikely that filtration into and flow through the SPS would contribute significantly to total flow across the entire GFB under these conditions. To determine whether the SPS has similar properties under normal physiological conditions, we have carried out further three-dimensional reconstruction of rat glomeruli perfused at physiologically normal hydrostatic and colloid osmotic pressures. These reconstructions show that the sub-podocyte space is even more restricted under these conditions, with a mean height of the SPS of 0.34µm, mean pathlength of 6.7±1.4µm, a mean width of the SPS exit pore of 0.15±0.05µm, and length of 0.25±0.05µm. Mathematical modeling of this sub-podocyte space based on a circular flow model predicts that the resistance of these dimensions is 2.47 times that of the glomerular filtration barrier and exquisitely sensitive to changes in the dimensions of the SEP, indicating that the SEP could be the principal regulator of the extravascular pressure in the SPS. This suggests a physiological role of the podocyte in the regulation of glomerular fluid flux across most of the glomerular filtration barrier.