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Plasma-derived lipid transfer protein (LTP) facilitates the transfer of cholesteryl ester and triglyceride between all lipoproteins. Kinetic models of the transfer event have suggested that transfer is facilitated through the physical interaction (binding) of LTP with its lipoprotein substrate. Such binding has been observed previously between LTP and high density lipoprotein (HDL), but not between LTP and low (LDL) or very low (VLDL) density lipoproteins. In the present study, the interaction of LTP with plasma lipoproteins has been re-evaluated. These experiments have employed Sepharose-bound lipoproteins in order to facilitate the rapid separation of unbound and lipoprotein-associated LTP. The validity of this approach in assessing LTP-lipoprotein interactions was evidenced by the fact that free (unbound) lipoproteins could competitively inhibit or disrupt the binding of LTP to the Sepharose-bound lipoproteins. LTP was observed to bind to VLDL, LDL, and HDL. Whereas VLDL- and LDL-LTP complexes were labile and almost completely dissociated in 90 min, HDL-LTP complexes remained intact during this time. Under equilibrium conditions, LTP binding to all lipoproteins was characterized by high affinity, saturable kinetics; the apparent affinities (Kd) of VLDL, LDL, and HDL for LTP were nearly the same (congruent to 25 nM). The results of two studies correlated lipid transfer activity with LTP binding to lipoproteins: 1) LTP binding and transfer activity increased in parallel as the amount of LTP in the assay was increased and 2) the inhibition of transfer activity caused by differing amounts of an inhibitory protein correlated with similar decrements in LTP binding. The latter data also suggest that the inhibitor protein suppresses lipid transfer activity by disrupting LTP-lipoprotein interactions. It is concluded that LTP avidly binds to VLDL, LDL, and HDL via a reversible, saturable mechanism and that the binding of LTP to the lipoprotein surface is an integral component of the lipid transfer reaction.