The extent to which the glomerular basement membrane (GBM) contributes to the charge selectivity of the glomerular capillary wall has been controversial. To reexamine this issue, the size and charge selectivity of filters made from isolated rat GBM were assessed, using polydisperse Ficoll and Ficoll sulfate as test macromolecules. Ficoll sulfate, a novel tracer with spherical shape synthesized for this purpose, exhibited little or no binding to serum albumin, thereby avoiding a major difficulty that has been reported with dextran sulfate. The sieving coefficients of Ficoll sulfate were not different from those of Ficoll at physiological ionic strength, although the values for Ficoll sulfate were depressed at low ionic strength. These results confirm that the GBM possesses fixed negative charges but suggest that its charge density is insufficient to confer significant charge selectivity under physiological conditions, where electrostatic interactions are relatively well screened. The sieving coefficients of Ficoll sulfate and Ficoll were elevated significantly and by similar amounts when bovine serum albumin (BSA) was present in the retentate at 4 g/dl. This could be explained as the combined effect of two nonspecific physical factors, namely, the reduction in filtration velocity due to the osmotic pressure of BSA and the effect on macromolecular partitioning of repulsive solute-solute interactions. The view that BSA does not affect the intrinsic properties of the GBM is supported also by the absence of an effect on the hydraulic permeability of isolated GBM. The sieving coefficient of BSA was roughly half that of Ficoll or Ficoll sulfate of similar Stokes-Einstein radius. Given the finding of negligible charge selectivity, this difference may be attributed to the nonspherical shape of albumin. The results suggest that, to the extent that isolated GBM is similar to GBM in vivo, the charge selectivity of the glomerular capillary wall must be due to the endothelial and/or epithelial cell layers.