We examined the effect of dialyzing oxidized low-density lipoprotein (oLDL) against Krebs-Ringer solution, in the absence (yielding d-oLDL) or presence (yielding EDTA-oLDL) of ethylenediamine tetraacetic acid (EDTA), to investigate the mechanism that underlies the inhibition of endothelium-dependent relaxation (EDR) by o-LDL. Oxidation of LDL by exposure to Cu2+ resulted in the formation of a thiobarbituric acid-reacting substance (TBARS) and lipid hydroperoxide (LPO). At a concentration of 5 mg/dl, d-oLDL markedly attenuated EDR in the porcine coronary artery. Analysis of d-oLDL by gel filtration revealed that TBARS was ditributed in both the lipoprotein and the aqueous phases, whereas LPO was present only in the lipoprotein particles. Lysophosphatidylcholine (LPC), which has been suggested to be responsible for the impairment of EDR by oLDL, was present not only in the lipoprotein but also in the aqueous phase. However, EDR inhibitory activity was observed only in the oLDL particles, not in the aqueous phase. Almost all Cu2+ associated with the oLDL particles was removed by dialysis of oLDL against Krebs-Ringer solution containing EDTA. EDTA-oLDL or native LDL, at concentrations as high as 75 mg/dl, exerted only a moderate inhibitory action on EDR, Both TBARS and LPO in EDTA-oLDL were distributed only in the lipoprotein particles, not in the aqueous phase. These results demonstrate that the impairment of EDR by oLDL is related both to LPO and to transition metal ions such as Cu2+ associated with the lipoprotein particles, not to the amount of the TBARS or negative charge, and that factors other than LPC may affect EDR.