The glomerular basement membrane (GBM) is an integral structural component of the glomerular filter, but its contribution to the hydraulic and macromolecular permeability properties of the glomerulus has been the subject of much controversy. We have modified previously reported methods to develop a technique with which to study filtration properties of microgram quantities of isolated GBM in vitro at physiological pressures. Rat glomeruli were sieved, and cells were removed with N-laurylsarcosine and DNase. GBM (150 micrograms; greater than 95% pure) were added to a mini-ultrafiltration cell and consolidated under pressure to form a continuous filter at the base of the cell. Water flux was identical to inulin clearance at applied pressures less than 150 mmHg and increased with progressive increments in the transmembrane pressure. Hydraulic conductivity of GBM was inversely related to the prevailing transmembrane pressure gradient. The hydraulic conductivity depended on albumin concentration in a manner that was not monotonic, with the conductivity being lower at 4 g/dl albumin than at 0 or 8 g/dl. When plasma was utilized as the retentate, the fractional clearance of albumin was over twice that of immunoglobulin G, and the fractional clearance of each protein was much higher than that in the intact glomerulus. On the basis of these results, both the hydraulic and macromolecular permeability of an individual layer of GBM are much greater than that reported for the intact glomerulus. The large quantitative differences between GBM permeability and that of intact glomeruli suggest a major contribution of cellular elements to glomerular permeability properties.