Graded dextrans have been used as tracers to identify the primary permeability barrier(s) to macromolecules among the structural elements (endothelium, mesangium, basement membrane, epithelium) of the glomerular capillary wall. Three narrow-range fractions of specified molecular weights and Einstein-Stokes radii (ESR) were prepared by gel filtration: (a) 32,000 mol wt, ESR = 38 Å; (b) 62,000 mol wt, ESR = 55 Å; and (c) 125,000 mol wt, ESR = 78 Å. These fractions are known to be extensively filtered, filtered in only small amounts, and largely retained, respectively, by the glomerular capillaries. Tracer solutions were infused i.v. into Wistar-Furth rats, and the left kidney was fixed after 5 min to 4 h. The preparations behaved as predicted: initially, all three fractions appeared in the urinary spaces, with 32,000 > 62,000 » 125,000. The smallest fraction was totally cleared from the blood and urinary spaces by 2.5 h, whereas the intermediate and largest fractions were retained in the circulation at high concentrations up to 4 h. With all fractions, when particles occurred in high concentration in the capillary lumina, they were present in similarly high concentrations in the endothelial fenestrae and inner (subendothelial) portions of the basement membrane, but there was a sharp drop in their concentration at this level—i.e., between the inner, looser portions of the basement membrane and its outer, more compact portions. With the two largest fractions, accumulation of particles occurred against the basement membrane in the mesangial regions with time. No accumulation was seen with any of the fractions in the epithelial slits or against the slit membranes. Dextran was also seen in phagosomes in mesangial cells, and in absorption droplets in the glomerular and proximal tubule epithelium. It is concluded that the basement membrane is the main glomerular permeability barrier to dextrans, and (since their behavior is known to be similar) to proteins of comparable dimensions (40,000–200,000 mol wt). The findings are discussed in relation to previous work using electron-opaque tracers to localize the glomerular permeability barrier and in relation to models proposed for the functions of the various glomerular structural elements.