Endothelial cells are covered with cell-born proteoglycans and glycoproteins. Blood plasma proteins, e.g. albumin, adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of endothelial cells to analyze interactions with the ESL. Albumin was spin-labeled with 5- or 12-DOXYL-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). Endothelial cell (EC) cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature regulated cavity of an ESR spectrometer. Alternatively, EC were labeled and then exposed to native albumin. At 37°C rotational correlation times () determined by modified saturation transfer ESR (ST-ESR) were 26 ns and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin. Presence of endothelial cells increased values for 5-DOXYL (to 37 ns) but not for 12-DOXYL. Albumin was able to completely take up the label from labeled EC within two minutes. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. The reduction in albumin surface mobility in the presence of EC at unchanged mobility of the protein proper and the fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between the albumin surface and the ESL but no rigid incorporation of albumin into a macromolecular network which would interfere with its transport function for poorly water soluble substances.