Co-adsorption kinetics of human low density lipoprotein (LDL) and serum albumin (HSA) on hydrophilic/hydrophobic gradient silica surface were studied using Total Internal Reflection Fluorescence (TIRF) and autoradiography. Two experimental systems were examined: (1) fluorescein-labeled LDL (FITC-LDL) adsorption from a FITC-LDL + HSA solution mixture onto the octadecyldi-methylsilyl (C18)-silica gradient surface, and (2) the FITC-LDL adsorption onto the HSA pre-adsorbed on the C18-silica gradient surface. Experiments with fluorescein-labeled albumin (FITC-HSA) and unlabeled LDL have been performed in parallel. The adsorption kinetics of FITC-LDL onto the hydrophilic silica was found to be transport-limited and not affected by co-adsorption of HSA. A slower adsorption kinetics of lipoprotein onto the silica with pre-adsorbed HSA layer resulted from a slow appearance of LDL binding sites exposed by the process of HSA desorption. In the region of increasing surface density of C18 groups, the FITC-LDL adsorption rate fell below the transport-limited adsorption rate, except in the very early adsorption times. Pre-adsorption of HSA onto the C18-silica gradient region resulted in a significant decrease of both the FITC-LDL adsorption rate and adsorbed amount. The lowest FITC-LDL adsorption was found in the region of C18 self-assembled monolayer, where the pre-adsorbed HSA layer almost completely eliminated lipoprotein binding.