This study was designed to determine whether modifications induced by dietary fats on the high-density lipoprotein3 (HDL3) physicochemical characteristics could affect cholesterol efflux and intracellular cholesterol content, leading to upregulation of low-density lipoprotein (LDL) receptor activity from cultured fibroblasts. Serum HDL3S were obtained from 12 healthy women aged 26 to 49 years who adhered to four 7-week isocaloric diets containing 30% of the caloric intake as fat. Of the total calories, 15.6% of each diet was provided by (1) milk fats, rich in saturated fatty acids; (2) sunflower oil, rich in n-6 polyunsaturated fatty acids; (3) olive oil, rich in monounsaturated fatty acids; and (4) rapeseed oil, rich in n-3 polyunsaturated fatty acids. HDL3 isolated after the monounsaturated fatty acid diet induced the greatest cellular [3H]free cholesterol efflux, reduced the content of intracellular cholesterol, and enhanced 125I-LDL degradation. Univariate regression analyses suggested that the increased capacity of HDL3 to promote cellular [3H]free cholesterol efflux was in part due to its greater fluidity, higher cholesteryl ester content, elevated linoleic to linolenic acid ratio in phospholipids, and its smaller size. In conclusion dietary fats induced physicochemical changes in HDL3, which strongly modulated cellular cholesterol homeostasis in vitro. These data also suggest a novel mechanism by which dietary fats exert their effect on atherosclerosis.