The present study documents the permeability characteristics of heart valvular endothelium to low-density lipoprotein (LDL), albumin, and horseradish peroxidase (HRP). Using quantitative autoradiography, LDL and albumin concentrations were measured within aortic valves of squirrel monkeys and rabbits after 30 minutes of in vivo circulation. The valvular concentration profiles were analyzed using theoretical mathematical models based on fundamental transport principles. In vivo transvalvular concentration profiles of LDL and albumin displayed the highest tissue concentrations immediately beneath the endothelium and displayed the lowest concentrations near the midline of the valve. Tissue concentrations of LDL and albumin displayed large differences in magnitudes between different regions of individual valve leaflets suggesting marked spatial variation in the permeability properties of the valvular endothelium to LDL and albumin; this was also seen visually with HRP. The results of the theoretical analysis showed that 1) the aortic valvular endothelium limits the uptake of LDL and albumin into the valvular tissue, 2) the permeability of the valvular endothelium differs widely from one region of a valve to another and even from one side of the valve to the other within a single valvular region, and 3) intramural diffusion is the predominant mode of transport for LDL and albumin within the aortic valve, even in valvular regions exposed to large pressure differences across the valve.