BACKGROUND 2 homogeneous assay kits, Determiner L HDL-C from Kyowa Medex, Co., Ltd. (Tokyo, Japan) (KWM) and Cholestest NHDL from Daiichi Pure Chemical, Co., Ltd. (Tokyo, Japan) (DIC), for HDL-C are widely used in laboratory tests worldwide. Meanwhile, it was reported that free cholesterol (FC) in HDL is not measured with one of these kits. We devised a novel evaluation method and analyzed the 2 kits in detail. METHODS Using HPLC, the residual amounts of cholesterol in reaction mixtures were compared before and after reaction with homogeneous reagents. Healthy sera were reacted with homogeneous reagents or with heated, non-enzyme reagents from each kit. The FC and total cholesterol in each lipoprotein were continuously measured with HPLC. RESULTS With KWM, the HDL-C was measured >95%.With DIC, all FC in sera was eliminated in the first reaction, and FC in HDL was not measured. Moreover, the peak X that was assumed to be a part of HDL was separated with DIC, and the cholesterol in the peak X was not measured. CONCLUSIONS 2 HDL-C assay kits were compared using a novel evaluation method. KWM was good overall. DIC was found to have 2 problems: 1) FC in HDL was not measured, and 2) the cholesterol in peak X that was assumed to be a part of HDL was not measured.