A novel monoclonal antibody (mAb), 8D3 (IgG2a), that specifically recognizes the murine transferrin receptor (TfR) was produced by immunizing a Lewis rat with a polyoma middle T oncogene-transformed endothelioma cell line. The 8D3 mAb was obtained by immunohistochemical screening for exclusive staining of vessels forming a blood-brain barrier (BBB), but not of other vessels. The anti-TfR mAb 8D3 recognizes the TfR also in FACS analysis and in western blots and should prove to be useful for affinity purification of the TfR. Whereas 8D3 brightly stains BBB-forming vessels in the central nervous system of mice, it does not stain the fenestrated capillaries within the choroid plexus and the circumventricular organs. In testis, where the blood-tissue barrier is located at the level of the Sertoli cells, the 8D3 mAb specifically stains Sertoli cells but not endothelial cells. Finally, in vitro, 8D3 does not interfere with iron uptake of lymphocytes as it does not influence their proliferation. Taken together, 8D3 represents a versatile new tool to study the tissue distribution of the murine TfR and TfR-mediated transcytosis across tissue barriers in the mouse.