Human HDL are heterogeneous in their metabolism and comprise small, nascent pre-beta-HDL and more mature alpha-HDL. Evidence exists that pre-beta 1-HDL is the initial acceptor of cellular free cholesterol, which then transfers sequentially to other pre-beta species and then, after esterification, into alpha-HDL. As HDL particles are themselves transformed during this process, we postulated that in disorders in which HDL-cholesterol is low, such as obesity, the distribution of HDL particles may be disturbed. In this study, we analyzed the HDL profile in 23 obese and 18 lean subjects, and further investigated the effects of dietary change in 15 obese subjects. HDL were separated by two-dimensional nondenaturing electrophoresis and the apoA-I content in each fraction was quantified. alpha 1-HDL in obese subjects was significantly lower (P < 0.001) and alpha 2-, alpha 3-, and pre-beta 1-HDL were significantly higher (P < 0.05 for alpha 2-HDL, P < 0.001 for alpha 3- and pre-beta 1-HDL) than in lean subjects. On stepwise regression analysis, body mass index accounted for 52% (negatively) of the variance in alpha 1-HDL and for 16% and 33% (positively) for the variances in alpha 3- and pre-beta 1-HDL, respectively. alpha 1- and pre-beta 3-HDL increased significantly after low-fat, oleic acid-rich, or alpha-linolenic acid-rich diets. The profile of alpha-HDL particles and also of pre-beta-HDL particles therefore shifted to smaller species in obese subjects, and this was influenced by dietary fat. Increased pre-beta 1-HDL-apoA-I in obese subjects is likely to derive from increased HDL catabolism but may also reflect diminished transformation of pre-beta 1- to pre-beta 2-HDL which might reduce capacity for reverse cholesterol transport and partly explain lower HDL-cholesterol levels.