According to the definition of the Lipid Research Clinic’s protocol, low-density lipoprotein (LDL) refers to the lipoprotein of density (d)=1.006–1.063 g/ml which contains another atherogenic lipoprotein, IDL (d=1.006–1.019 g/ml). Because metabolic properties are largely different between LDL and IDL, LDL is now defined as the lipoprotein of d=1.019–1.063 g/ml. Recently direct LDL-cholesterol assay kits using novel surfactants (the homogeneous methods) have become commercially available and widely used in Japan. The aim of this study is to examine how three direct LDL-cholesterol assay kits, LDL-EX, Choletest-LDL and Determinor-L LDL, react with pure LDL (d=1.019–1.063 g/ml) and IDL (1.006–1.019 g/ml) fractions isolated by ultracentrifugation. Thirty-one healthy subjects and one type III dysbetalipoproteinemic patient were enrolled in this study. All homogeneous methods highly correlated with LDL-cholesterol (r=0.95–0.98), although the values for LDL-EX were closer to the values for ultracentrifugation than were those of the other two methods (95 vs. 86–87%, P<0.0001). Cross-reactivity with IDL was 31, 47 and 64% for LDL-EX, Choletest-LDL, and Determinor-L LDL, respectively. Similar results were obtained in the IDL from a type III dysbetalipoproteinemic patient. These results suggest that LDL-cholesterol measured by LDL-EX better reflects pure LDL fraction with weaker cross-reaction with IDL than other homogeneous methods.