Recent publications questioned the validity of endothelial cell (EC) culture studies of glycocalyx (GCX) function, due to findings that GCX in vitro may be substantially thinner than in vivo. The assessment of thickness differences is complicated by GCX collapse during dehydration for traditional electron microscopy. We measured in vitro GCX thickness using rapid freezing/freeze substitution transmission electron microscopy (RF/FS-TEM), taking advantage of high spatial resolution provided by TEM and the capability to stably preserve the GCX in its hydrated configuration by RF/FS. Bovine aortic and rat fat pad endothelial cells (BAEC and RFPEC) were subjected to conventional- or RF/FS-TEM. Conventionally preserved BAEC GCX was ∼0.040 μm in thickness. RF/FS-TEM revealed impressively thick BAEC GCX of ∼11 μm and RFPEC GCX of ∼5 μm. RF/FS-TEM also discerned GCX structure and thickness variations due to heparinase III enzyme treatment and extracellular protein removal, respectively. Immunoconfocal studies confirmed that the in vitro GCX is several microns thick and is comprised of extensive and well integrated heparan sulfate, hyaluronic acid, and protein layers. New observations by RF/FS-TEM reveal substantial GCX layers on cultured EC, supporting their continued use for fundamental studies of GCX and its function in the vasculature.