Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Δ53−71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k_a, but different dissociation rate constants, k_d, that depended on the presence of apoA-I (k_d decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k_a for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Δ53−71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein−protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.