BACKGROUND The use of high-density lipoprotein cholesterol (HDL-C) levels as a risk factor for coronary heart disease necessitates an accurate and precise method for measuring HDL-C. The Centers for Disease Control and Prevention HDL-C reference method (RM) and designated comparison method (DCM) are time-consuming, expensive, and impractical for routine clinical use. We evaluated the Liquid N-geneous (LN-gen) HDL-C assay (Genzyme Diagnostics, Cambridge, Mass) to determine if this homogeneous reagent meets the National Cholesterol Education Program requirements for HDL-C evaluation. METHODS Accuracy of the LN-gen HDL-C assay was compared in combination with phosphotungstic acid (PTA) precipitation with DCM HDL-C for normotriglyceridemic serum specimens (triglycerides < 2.0 g/L) and with RM HDL-C for specimens with triglycerides levels > or = 2.0 g/L. METHODS Genzyme Diagnostics (with RM and DCM assayed by Pacific BioMetrics Inc, Seattle, Wash) and the Lipid Reference Laboratory of the University Hospital Rotterdam, The Netherlands. RESULTS Linear regression to DCM (n = 90) was (LN-gen = 1.015 DCM + 0.01 g/L, r = 0.993, SE = 0.015 g/L) and (PTA = 1.004 DCM - 0.017 g/L, r = 0.980, SE = 0.025 g/L), with a mean percent bias to DCM of 3.3% and -2.8% for LN-gen and PTA, respectively. The comparison with RM (n = 69) showed an increased mean bias for PTA (-5.8%) as compared with LN-gen (1.5%). The correlation and regression equations were (LN-gen = 1.020 RM - 0.002 g/L, r = 0.985, SE = 0.017 g/L) and (PTA = 1.042 RM - 0.032 g/L, r = 0.984, SE = 0.018 g/L). The precision of LN-gen was confirmed at < 2.1% coefficient of variation, and the total error was calculated to be < or = 7.7% for both normotriglyceride and elevated triglyceride specimens at HDL-C decision points of 0.35 g/L and 0.60 g/L. CONCLUSIONS The LN-gen HDL-C assay offers a cost-effective convenient method for meeting the 1998 precision, bias, and total error recommendations of the National Cholesterol Education Program.