Lipid transfer inhibitor protein (LTIP) regulates cholesteryl ester transfer protein (CETP) activity by selectively impeding lipid transfer events involving low density lipoproteins (LDLs). We previously demonstrated that LTIP activity is suppressed in a dose-dependent manner by sodium oleate and that its activity can be blocked by physiological levels of free fatty acids [R.E. Morton, D. J. Greene, Arterioscler. Thromb. Vasc. Biol. 17 (1997)]. These data further suggested that palmitate has greater LTIP suppressive activity than oleate. In this report we define the ability of the major non-esterified fatty acids (NEFAs) in plasma to modulate LTIP activity. The greater suppression of LTIP activity by palmitate compared to oleate noted above was also seen in lipid transfer assays with various lipoprotein substrates and in the presence of albumin, showing that the relative effects of these two NEFAs are independent of assay conditions. To assess the effect of other NEFAs on LTIP activity, pure NEFAs were added to assays containing (3)H-cholesteryl ester labeled LDLs, unlabeled high density lipoproteins (HDLs) and CETP+/-LTIP. Whereas myristate, palmitate, stearate, oleate and linoleate stimulated CETP activity to varying extents, all NEFAs suppressed LTIP activity. Among these NEFAs, LTIP suppressive activity was greatest for the long-chain saturated and monounsaturated NEFAs. In contrast, linoleate and myristate were poor inhibitors of LTIP activity. The effects of increasing amounts of a given NEFA on LTIP activity correlated well with the increase in LDL negative charge induced by that NEFA, yet this relationship was unique for each NEFA, especially stearate. Notably, as measured by fluorescence anisotropy, the suppression of LTIP was highly and negatively correlated with the decreased order in the molecular packing of lipoprotein surface phospholipids caused by all NEFAs. Long-chain, saturated and monounsaturated NEFAs appear to be most effective in this regard partly because of their preferential association with LDLs where LTIP inhibition likely takes place. We hypothesize that NEFAs suppress LTIP activity by perturbing the surface properties of LDLs and counteracting the heightened molecular packing normally caused by LTIP. Diets rich in long-chain saturated and monounsaturated fatty acids may lead to a greater suppression of LTIP activity in vivo, which would allow LDLs to participate more actively in CETP-mediated lipid transfer reactions.