The present study aims to experimentally elucidate subtle structural features of the rat valve leaflet and the related nature of macromolecular transport across its endothelium and in its subendothelial space, information necessary to construct a rational theoretical model that can explain observation. After intravenous injection of horseradish peroxidase (HRP), we perfusion-fixed the aortic valve of normal Sprague-Dawley rats and found under light microscopy that HRP leaked through the leaflet's endothelium at very few localized brown spots, rather than uniformly. These spots grew nearly as rapidly with HRP circulation time prior to sacrifice as aortic spots, particularly when the time axis only considers the time the valve was closed. These results suggest that macromolecular transport in heart valves depends not only on the direction normal but also parallel to the endothelial surface and that convection, as well as molecular diffusion, plays an important role in macromolecular transport in heart valves. Transmission electron microscopy of traverse leaflet sections after 4 minutes HRP circulation showed a very thin (~150 nm), sparse layer immediately beneath the endothelium where HRP concentration was much higher than that in the matrix below it. Nievelstein-Post et al.'s (18) ultra-rapid freezing/rotary shadow etching of the normal rabbit valve's subendothelial space supports the existence of this very thin, very sparse "valvular subendothelial intima," in analogy to the vascular subendothelial intima.