We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of malondialdehyde-modified low-density lipoprotein (MDA-LDL) in human serum. A monoclonal antibody against MDA-LDL (ML25) used in our method recognized not only MDA-LDL but also other MDA-modified proteins. However, MDA-LDL was able to be detected specifically by using a combination of ML25 and an antibody specific for apolipoprotein B (apo B) (AB16), which was conjugated with beta-galactosidase. Using this method, measurable amounts of MDA-LDL were detected in the sera of 40 healthy individuals. MDA-LDL was observed to be mainly distributed in the human LDL fraction separated by density gradient ultracentrifugation, while in each lipoprotein subfraction the largest amount of MDA-LDL per protein was found at a subfraction between LDL and HDL. The particle size of LDL in this fraction was smaller than that of LDL in the main LDL fraction, as assessed by electrophoresis. In addition, LDL oxidized by Cu2+ was also detectable with this method. We conclude that our method is sensitive and specific for MDA-LDL and might be useful for investigating MDA-LDL in the human circulation.