The nonspecific lipid-transfer protein (nsL-TP) from bovine liver was studied by measuring the binding and transfer of the fluorescent phospholipid 1-palmitoyl-2-[6-(1-pyrenyl)-hexanoyl]-sn-glycero-3-phosphocholine (PamPryGroPCho). A kinetic model is presented involving three steps: (a) interaction of nsL-TP with a membrane surface; (b) equilibration of PamPyrGroPCho monomers between the membrane and nsL-TP; and (c) dissociation of the nsL-TP/PamPyrGroPCho complex from the membrane surface. Steady-state analysis of the model yielded theoretical equations describing both binding and transfer kinetics. Computer analysis, using these equations, showed good fits with the experimental results and several kinetic constants could be calculated. From these constants it was inferred that incorporation of acidic phospholipids into vesicles enhanced the interaction of nsL-TP with the membrane interface (step a), without affecting the equilibrium binding of phospholipid monomers to nsL-TP (step b). As a result, the rate of nsL-TP-mediated PamPyrGroPCho transfer from donor to acceptor vesicles was greatly affected. Under the conditions of incubation, incorporation of the acidic lipids in the donor membrane vesicles stimulated transfer, whereas incorporation of these lipids in the acceptor membranes could lead to a virtually complete inhibition of transfer. From the results it is concluded that the formation of a soluble lipid-nsL-TP complex is the key step in nsL-TP-mediated phospholipid transfer.