In order to elucidate the relations between the amount of surface functional groups and interaction forces, BSA adsorption experiments were performed, using highly carboxylated PS/PMAA microspheres as well as conventional ones. Two kinds of interaction forces were considered in this study, hydrogen bonding and hydrophobic interactions, while ionic interactions were assumed to be small or constant, Hydrophobic interactions were dominant in the low N-c (number density of surface carboxyl groups) region, below 1 carboxyl group nm(-2), and were relatively sensitive to pH, while hydrogen bonding was dominant in the high N-c region, above 2 carboxyl groups nm(-2), irrespective of pH, The transition region between these two interaction forces was 1 similar to 2 carboxyl groups nm(-2) on the surface of a microsphere, When comparing the two extremes of hydrophobic interaction and hydrogen bonding, the latter was stronger, which was different from earlier studies, It can be explained that the conventional carboxylated microspheres do not have enough carboxyl groups on their surfaces, Adsorption constant K diminished as N-c increased, which means that the affinity itself is reduced by hydrogen bonding, Adsorbed layer thicknesses calculated from the C-m (adsorbed amount in equilibrium) were 4.66 similar to 7.08 nm; this means that the BSA molecules exist between the side-on and end-on mode.