Many aspects of the glomerular filtration of macromolecules remain controversial, including the location of the major filtration barrier, the effects of electrical charge, and why the filtration barrier does not clog. We examined these issues in anesthetized Necturus maculosus, using fluorescently labeled probes and a two-photon microscope. With the high resolution of this system and the extraordinary width (~3.5 microns) of the glomerular basement membrane (GBM) in this salamander, we were able to visualize fluorescent molecules in the GBM in vivo. GBM/plasma concentration ratios for myoglobin, ovalbumin, and serum albumin did not differ from that of inulin, indicating that the GBM does not discriminate among these molecules. The GBM/plasma concentration ratios for fluoresceinated dextrans 500 kDa and 2,000 kDa were significantly below that of inulin. Glomerular sieving coefficients (GSCs) for various macromolecules decreased as molecular weight increased, and the GSCs for bovine or human serum albumin were extremely low. The effect of electrical charge on filterability of a macromolecule was also examined. The GSCs for native (anionic) and neutral human serum albumin were not significantly different, nor did GSCs for anionic and neutral 40 kDa dextrans differ, indicating that charge has no detectable effect on filterability of these macromolecules. These studies indicate that the main filtration barrier to albumin is the podocyte slit diaphragm. Electron microscope studies revealed many cell processes within the GBM. Macromolecules that penetrated the GBM were taken up by mesangial cells and endothelial cells, suggesting that these cells help to prevent clogging of the filter.