In recent years, the endothelial cell surface glycocalyx has emerged as a structure of fundamental importance to a broad range of phenomena that determine cardiovascular health and disease. This new understanding of the functional significance of the glycocalyx has been made possible through recently developed experimental techniques using intravital microscopy that are capable of directly probing the glycocalyx in vivo. Using fluorescent microparticle image velocimetry in venules and endothelialized cylindrical collagen microchannels, we show that the hydrodynamically relevant endothelial cell glycocalyx surface layer observed in microvessels in vivo (0.52±0.28 μm thickness), which is a fundamental determinant of the hydrodynamic and mechanical environment at the endothelial cell surface, is absent from human umbilical vein (0.03±0.04 μm thickness) and bovine aortic (0.02±0.04 μm thickness) endothelial cells grown and maintained under standard cell culture conditions in vitro. An endothelial surface–bound glycosaminoglycan layer, not necessarily indicative of but having similar hydrodynamic properties to the endothelial glycocalyx observed in vivo, was detected (0.21±0.27 μm thickness) only after hyaluronan and chondroitin sulfate were added to the cell culture media at hyperphysiological concentrations (0.2 mg/mL perfused for 75 minutes). The implications of this glycocalyx deficiency under standard cell culture conditions in these pervasive in vitro models broadly impact a myriad of studies involving endothelial cell monolayers in which inferences are made that may depend on endothelial cell surface chemistry. In light of these findings, conclusions drawn from such studies in the areas of microvascular permeability, inflammation, mechanotransduction, and atherosclerosis must be carefully reconsidered.