Numerous studies have indicated that oxidative modification of low-density lipoprotein(LDL) plays a critical role in the pathogenesis of atherosclerosis. Malondialdehyde-modified LDL(MDA-LDL) is one of the candidates which is the oxidative product of LDL. However, the existence of MDA-LDL in the circulation has been in dispute. Therefore, for the assessment of oxidized-LDL in human serum, we developed a sensitive enzyme-linked-immunosorbent assay(ELISA) for the detection of MDA-LDL. In our method, monoclonal antibody against MDA-LDL, ML25 was used. ML25 recognized MDA-LDL as well as MDA-modified proteins by a solid-phase competitive enzyme immunoassay. Therefore, to establish an ELISA method which is specific for detection of MDA-LDL, ML25 was combined with apoB-specific antibody (AB16) as the second antibody. Using this method, MDA-LDL was detectable in the sera of 314 healthy individuals. The concentration of MDA-LDL preferably ranged from 20 to 80 units/l when the absorbance with artificially prepared MDA-LDL at the concentration of 1 mg/l was defined as 1 unit/l. Furthermore, assays for lipoprotein subfractions separated by density-gradient ultracentrifugation revealed that MDA-LDL was mainly distributed in the LDL fraction as expected, and MDA-LDL/apoB ratio showed a peak at small, dense LDL fractions. This finding seems to be quite interesting since an elevated level of small, dense LDL has been reported to be associated with an increased risk of atherosclerosis. We concluded that our method is useful for specific detection of MDA-LDL in human serum and might be an elevation method for atherogenicity.