We monitored the interactions between pairs of molecules where the antigen adenylate kinase (AK) was immobilized on the surface of a chip and the antibody against AK, McAb3D3, was in solution. The association data that we obtained were not always accurately described by the expected pseudo-first-order reaction mechanism. A better description of the association data was achieved with a double-exponential function. Various models were applied to describe these observations: mass transport-controlled processes, inhomogeneous immobilized ligands, or inhomogeneous soluble analytes. Inhomogeneous immobilized ligands seemed to be the most likely explanation for the observed biphasic association kinetics. We simulated the kinetics of the SPR signal under the above-mentioned conditions. Plots of dR/dt versus R of the association phase showed characteristic differences between those nonlinearities resulting from mass transport limitation and those from inhomogeneous ligands. The plots of dR/dt versus R of McAb3D3 binding to immobilized AK show positive curvatures, indicating that the observed biphasic association kinetics is due to the inhomogeneity of the immobilized ligands. This is consistent with the results obtained from the comparison of various model fittings.