To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton‐Hunter reagent,125I‐sulfosuccinimidyl (hydroxyphenyl) propionate (125I‐s‐SHPP). This technique led to a 10‐fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase‐catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet‐to‐dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of125I‐s‐SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin‐converting enzyme and β2‐microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiological stimuli.— Merker, M. P.; Carley, W. W.; Gillis, C. N. Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung.FASEB J.4: 3040‐3048; 1990.