This work describes the binding of carbonic anhydrase (CA) to mixed self-assembled monolayers (SAMs) presenting benzenesulfonamide ligands at a surface consisting primarily of tri(ethylene glycol) [(EG)(3)OH] groups. Surface plasmon resonance (SPR) quantified the binding of CB to the benzenesulfonamide groups. Two factors influenced the binding of CA: (a) the density of benzenesulfonamide groups at the surface, and (b) the coverage of the surface with molecules of CA adsorbed to these benzenesulfonamide groups. At low mole-fractions of benzenesulfonamide groups in the mixed SAM where the binding of CA is highly (>90%) reversible, we observe. (a an approximately 10-fold decrease in the observed bimolecular rate constant for association, k(on,obs), during the binding of CA(as the fraction of the surface covered by adsorbed CA increases from similar to 0.15 to similar to 0.35, the value of k(on,obs) decreases from -40 x 10(3) M-1 s(-1) to -4 x 10(3) M-1 s(-1)); ib) almost no corresponding changes in the observed unimolecular rate constant for dissociation (k(off,obs) similar to 0.005 s(-1)) during the dissociation of CA from the surface. These observations establish that k(on,obs) is influenced by the extent of coverage of the surface with CA, but that k(off,obs) is not. At low surface densities of arylsulfonamide groups, one hypothesis that rationalizes these data is that the decrease in k(on,obs) reflects repulsive steric interactions between molecules of CA. near the surface and those already adsorbed. Each molecule of biospecifically adsorbed CA shields proximal benzenesulfonamide ligands from binding to incoming molecules of CA, and decreases the surface density of these ligands that are accessible to CA, at a rate that increases nonlinearly with the quantity of CA already adsorbed.