The passage of various endogenous proteins [such as albumin, transferrin, immunoglobulin G (IgG), and immunoglobulin M (IgM)] across GBM was studied in vivo in normal Munich-Wistar rats. Glomeruli were fixed by three different methods: in situ drip-fixation, perfusion- and immersion-fixation; then they were processed for immunogold electron microscopy. The most reproducible results were obtained with in situ drip-fixation. Albumin, transferrin and IgG penetrated into GBM, but IgM did not. Morphometry revealed that density of albumin increased towards the inner 1/5 to 1/3 of GBM (junction of lamina rara interna and lamina densa) and decreased towards the subepithelial region of GBM, whereas density of IgG and transferrin was the highest at the subendothelial site and declined towards the subepithelial side of GBM. These findings suggest that central and/or outer zone of GBM constitute the main filtration barrier for albumin, and that subendothelial zone may contribute also to the charge-selective barrier. It is also suggested that the subendothelial zone acts more effectively as a filtration barrier for IgG and transferrin than for albumin. In the outer zone of GBM, which roughly corresponds to lamina rara externa visualized by conventional electron microscopy, the relative density of IgG and transferrin was higher than that of albumin. Since the pI of albumin was lower than that of IgG and transferrin, this finding suggests that subepithelial zone of GBM also acts as a charge-selective barrier. In conclusion, the main GBM filtration barrier for albumin might be the central and outer zones of GBM, and that for transferrin and IgG might be the entire width of GBM.(ABSTRACT TRUNCATED AT 250 WORDS)