Polydisperse Ficoll mixtures have been used to explore glomerular sieving. Ficoll appears to be neither absorbed nor secreted by the renal tubule, and so urinary Ficoll concentrations reflect only the glomerular filtration barrier. The literature is contradictory regarding Ficoll's behavior as an idealized spherical solute. Further definition of Ficoll transport will inform interpretation of in vivo results. Flat-sheet membranes comprising a uniform array of slit pores measuring 8 nm by 45 mum were perfused with FITC-labeled Ficoll 70 and BSA. Ficoll and BSA concentrations were quantified by gel-permeation chromatography and Bradford assay, respectively. BSA and Ficoll molecules with diameters equal to approximately half of the slit pore width displayed hindered transport in agreement with modeled rigid sphere transport through slit-shaped pores. Ficoll molecules larger than approximately 0.65 slit width displayed transport rates in excess of predictions. Ficoll molecules with Stokes-Einstein diameters greater than the pore dimension were observed in permeate samples. We present data for Ficoll filtration through a novel array of well-defined pores, which illustrate that Ficoll is well modeled as an ideal sphere in one size domain, but the model breaks down as molecular diameter approaches pore size. These data inform the present debate regarding glomerular filtration and affect conclusions drawn from the use of Ficoll as a tracer molecule. The apparent hyperpermeability of Ficoll through slit-shaped pores suggests that further modeling incorporating deformation of the molecule is necessary when using Ficoll solutions to characterize membranes.