Three-dimensional organization of vesicles was examined to elucidate the transendothelial transport properties for macromolecules in rat aortic endothelium using ultrathin serial sections and horseradish peroxidase (HRP) as a tracer molecule. A total number of reconstructing vesicles was 1,298 in nine series electron micrographs, if each vesicular entity was counted as one regardless of composite number of vesicles. The vesicles could be classified into the following six types: HRP-positive luminal, abluminal and intercellular invaginations; HRP-positive channels; HRP-positive and -negative vesicles. The vesicular invaginations and the channels occupied 97.8% and 0.9% of the total vesicles, whereas HRP-positive and -negative free vesicles were found in 0.8% and 0.5%, respectively. The average numerical density of the luminal invagination was 41.1/μm2 and approximately equal to that of the abluminal invagination (42.0/μm2), whereas the frequency of the latter was 1.4 times higher than that of the former since the abluminal surface of the endothelium was more irregular to increase a surface area. Each endothelial region varied in the vesicular density and the peripheral region generally showed the higher density, although the transendothelial channels composed of vesicles were not always found in every peripheral region. These results suggest that the shuttle hypothesis is unsuitable to explain “vesicular transport” in the arterial endothelium as well as in the capillary endothelium and that the channels in the peripheral region mainly control transendothelial transport for macromolecules via vesicles. © 1993 Wiley-Liss, Inc.