Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC A ) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC A . Multiple factors influenced GSC A , including the kidney depth of image acquisition (10-20 mm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC A in Simonsen Munich Wistar rats from 0.03560.005 to 0.01660.004 (P,0.01). Frömter Munich Wistar rats had a much lower GSC A in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability. Over the past several years, the roles the glomerular filtration barrier and the proximal tubule play in the development of albuminuria have been debated. 1,2 The present textbook model, in which albumin filtration across a normal glomerulus is thought to be minimal, has recently been challenged. A wide array of genetic, molecular, biochemical, and imaging studies are consistent with proximal tubule cells (PTCs) having a role in reclaiming filtered proteins, including albumin, and thus minimizing proteinuria. These studies include the following: knockout mice lacking Na+-H+ exchanger isoform 3 or chloride channel-5; 3 megalin-cubilin complex defects; 4 Rab 38-mediated tubular proteinuria and albuminuria; 5 statin-mediated inhibition of guanosine triphosphatase prenylation with reduced proximal tubule (PT) endocytosis; 4,6-8 mice lacking FcRn, the IgG and albumin receptor; 9,10 and selective PTC injury using D-serine 11 or the selective expression of diphtheria toxin receptor on PTC. 12 In a preliminary communication, Menzel and colleagues showed that PT reabsorption and transcytosis of podocyte produced and secreted albumin labeled with V5 and hemagglutinin tags. 13 Finally, recently published data 14 using advanced scanning electron microscope imaging techniques indicate that the podocyte slit diaphragm has pores that are much larger than previously determined and are of the size required for albumin filtration.The development of in vivo two-photon microscopy has enabled the direct visualization and quantitation of fluorescent compounds as they filter through the glomerulus and are endocytosed by