Background/Aims: The vasculature has become an important target in the development of therapies for increasing numbers of human diseases, yet there are few reliable markers available to identify the endothelium in rodent models. We have characterized the expression, subcellular localization and accessibility of the rat pan-endothelial marker podocalyxin (podxl) using a newly developed monoclonal antibody (mAb), G278. Methods: podxl expression and accessibility to binding by G278 were determined in the rat by a variety of experimental approaches. Results: mAb G278 reliably immunostained blood vessels of all types and of every size in fresh-frozen, fixed-frozen and paraffin-embedded sections of all tissues, but did not stain lymphatic vessels. Western blotting, in vivo imaging and biodistribution analyses demonstrated that the highest levels of endothelial podxl were found in the lung and heart. We also determined that podxl is not enriched in caveolae and that its expression can be modulated in the tumor microenvironment. Conclusion: Our study shows that podxl is a better identifier of rat endothelia than are some of the more commonly used markers and that mAb G278 is a robust antibody for use not only in identifying rat blood vessels but also as a tool to elucidate podxl function.