The present study analyzed serum paraoxonase 1 (PON1) distribution among HDL and lipoprotein-deficient serum (LPDS) in atherosclerotic patients, and compared PON1 biological functions in these fractions. Serum HDL and LPDS fractions were isolated from control healthy subjects, diabetic and hypercholesterolemic patients. PON1 activities and protein in HDL/LPDS, as well as its ability to protect against lipid peroxidation and to stimulate HDL/LPDS-mediated macrophage cholesterol efflux were measured. In LPDS from controls, PON1 protein and a significant paraoxonase activity were found, whereas arylesterase and lactonase activities were substantially reduced compared to HDL, by 78% and 88%, respectively. In diabetic patients, PON1 protein and paraoxonase activity in HDL were significantly decreased by 2.8- and 1.7-fold, respectively, compared with controls' HDL. In parallel, in these patient's LPDS, PON1 protein and paraoxonase activity were markedly increased by 3.7- and 1.7-fold, respectively, compared with controls' LPDS. PON1 in HDL (but not PON1 in LPDS) significantly decreased AAPH-induced lipid peroxides formation by 33%, and increased macrophage cholesterol efflux by 31%. We conclude that PON1 is less antiatherogenic when present in LPDS than in HDL. The abnormal serum PON1 distribution in diabetic patients, could be responsible for the accelerated atherosclerosis development in these patients.