The development of renal histo-architecture substantially depends on the three-dimensional extension of the collecting duct (CD) ampulla, since under its influence, nephron induction takes place in the surrounding mesenchyme. Recently, micro-fibers were detected by soybean agglutinin (SBA), which line from the basal aspect of each CD ampulla through the mesenchyme towards the organ capsule in embryonic kidney. Their unique distribution suggests that they may play an important role in the control of CD ampulla growth and in forming the renal stem cell niche. A profound analysis of interstitial proteins between the CD ampulla and the nephrogenic mesenchyme is lacking. Consequently, the goal of the current investigation was to colocalize the micro-fibers detected by SBA with interstitial proteins. For this reason a detailed cell biological analysis of extracellular molecules at this site was carried out. Double labeling showed that the micro-fibers do not correspond to known collagens and other extracellular matrix molecules such as agrin, versican or MMP-9. In addition, it could be demonstrated that the micro-fibers do not contain epithelial or mesenchymal cell elements. Furthermore, two-dimensional electrophoresis with subsequent Western blotting yielded two different amino acid sequences (1: GHYADPTSPR; 2: NNGCCSSDYHA) obtained from SBA-labeled protein spots. Both amino acid sequences could not be assigned to known rodent proteins. The findings suggest that the SBA-labeled micro-fibers represent a new type of extracellular structure between the CD ampulla, the mesenchyme and the organ capsule.