Unlike pretreatment with native low density lipoproteins (LDL), pretreatment of rabbit aortic segments with oxidized LDL reduced significantly the acetylcholine-mediated maximal relaxation. The absolute difference between maximal relaxation values obtained with oxidized LDL and native LDL correlated significantly with the formation of 7-ketocholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol. When added individually, 7-ketocholesterol and 7β-hydroxycholesterol reduced maximal relaxation in a time- and concentration-dependent manner. Complementary studies with cultured human umbilical vein endothelial cells (HUVEC) were conducted in order to determine whether the inhibitory effect of 7-ketocholesterol could relate to the reduction of the release of nitric oxide (NO) which was determined by electrochemical detection using a nickel porphyrin- and Nafion-coated carbon microfiber electrode. In the presence of the antioxidant butylated hydroxytoluene (BHT), 7-ketocholesterol, but not cholesterol was able to significantly reduce the histamine-mediated release of NO from HUVEC in a time- and concentration-dependent manner. This inhibitory effect was independent of the known cytotoxicity of 7-ketocholesterol, and under the experimental conditions used in the present study neither the morphology, nor the viability of endothelial cells was affected by 7-ketocholesterol treatments. It is concluded that some cholesterol oxides, in particular 7-ketocholesterol can specifically inhibit the release of NO from endothelium cells, an observation that might account at least in part for the ability of oxidized LDL to impair the endothelium-dependent relaxation of arteries.