We used BlAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD8 I LEL-MBP and the El E2, E2 or E2661 HCV envelope proteins were similar. However, the dissociation rate constants of CD81 LEL-MBP were higher than those of CD81 LEL-GST. Interestingly, the dissociation rate constant of CD8]LEL-GST from E1E2 was much lower than from E2 or E2661. The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate. (C) 2005 Elsevier Inc. All rights reserved.