HDL becomes enriched with non-esterified fatty acids (NEFAs) in some pathologies, such as nephrotic syndrome, as well as after aerobic exercise. However, little is known about the impact of NEFAs on HDL metabolism. We investigated the effects of one NEFA, the palmitic acid, on HDL structure and catabolism.HDL enrichment with palmitic acid (HDL Pal ) was performed by fusing phosphatidyl choline small unilamellar vesicles containing the NEFA with human HDL isolated from a pool of 5 normolipidemic plasma. HDL enriched only with phosphatidyl choline (HDL Phl ) and native HDL (HDL Ctrl ) were included as controls.As expected, HDL Pal surface charge density was higher than HDL Phl and HDL Ctrl (2014.4±164.8 vs. 1682.7±149.5 and 1758.2±124.3-esu/cm 2 , respectively, p<0.05). Both, HDL Pal and HDL Phl were better substrates for cholesteryl esters transfer protein (CETP) than HDL Ctrl (% of transfer, 13.02±3.8 and 12.7±4.5 vs. 7.8±2.7% in 16 h, respectively, p<0.05). HDL Pal apo A-I catabolism in vivo, as performed in New Zealand white rabbits by exogenous radiolabeling, was markedly lower than that of HDL Phl and HDL Ctrl (fractional catabolic rate, 0.019±0.008 vs. 0.030±0.005 and 0.047±0.003 h −1 , respectively, p<0.001), suggesting that negative charge is inversely related to HDL-apo A-I catabolism.Enrichment with palmitic acid increases the negative electric charge of HDL at physiological pH, contributes to decrease their catabolism, and is associated to an enhanced lipid transfer by CETP that has been related to the atherogenic process.