Low-density lipoprotein (LDL) cholesterol can not be calculated from other lipid measurements when samples are obtained from nonfasting individuals or when triglycerides are > or = 4.0 g/L. We have evaluated a direct LDL cholesterol assay for analyzing 115 fresh serum samples obtained from fasting and nonfasting dyslipidemic patients with triglycerides < or = 35.85 g/L, who were receiving diet and (or) drug treatments. Results were highly correlated with those by ultracentrifugation (r = 0.97), with a mean/median bias of -2.9%/0.7% (-0.001/0.010 g/L) and an absolute bias of 9.5%/6.4% (0.119/0.090 g/L). The assay correctly classified LDL cholesterol concentrations < 1.30 g/L 81% of the time, 1.30-1.60 g/L 76% of the time, and > or = 1.60 g/L 94% of the time. Precision studies provided within- and between-run CVs in the range of 1.2-3.8% and 2.0-5.1%, respectively. Our data indicate that this assay is an accurate method for measuring LDLC directly from fresh serum obtained from fasting or nonfasting subjects with a wide range of triglyceride values.