To assess the effects of oxidative modification, human HDL was oxidised in vitro for 12 h (Ox-HDL12) and 24 h (Ox-HDL24) under similar conditions to those commonly used for LDL. The procedure resulted in: an increase in thiobarbituric acid reactive substances but with marginal change in electronegativity; protein denaturation accounting for 16% and 45% loss of immunoreactive apoprotein A-I in the Ox-HDL12 and Ox-HDL24 respectively relative to the non-oxidised, native HDL (Nat-HDL); a decrease in the polyunsaturated fatty acids of the triglyceride, cholesterol ester and phospholipid components of the lipoprotein; an increase in the proportion of short chain saturated fatty acids while the monounsaturated fatty acids remained relatively unchanged. Studies with human macrophages demonstrated: a decrease of 16% and 30% in the capacity of the Ox-HDL12 and Ox-HDL24 respectively to efflux intracellular free cholesterol; 125I-Ox-HDL24 uptake and degradation was directly comparable with that of 125I-Ac-LDL; the addition of excess unlabelled Ox-HDL24, Ac-LDL, Ox-LDL24 and Nat-HDL resulted in 74%, 67%, 69% and 19% displacement of the 125I-Ox-HDL24 respectively; fucoidin and dextran sulphate displaced 125I-Ox-HDL by 20% and 40% respectively; intracellular free and esterified cholesterol was increased 2.5-fold and 4-fold respectively relative to Nat-HDL on incubation with Ox-HDL24. These findings suggest that HDL is susceptible to oxidative modification leading to recognition by the scavenger receptor of macrophages and subsequent intracellular cholesterol accumulation. As such, the in vivo protective role of HDL in cardiovascular disease can be reversed in those circumstances in which HDL, like LDL, undergoes oxidative modification.