Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger arterial disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. Here we visualised pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighbouring cells, high-density-lipoprotein-sized tracers crossed at tricelluar junctions whilst low-density-lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimised shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimised transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimise transverse shear, supporting its postulated pro-atherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo.