Heightened awareness of the importance of cholesterol and heart disease has increased cholesterol testing in the United States. The demand for reliable cholesterol measurements has become a focal concern of the patient as well as the clinician. This paper covers the major analytical and preanalytical issues and factors that can affect the reliability of cholesterol results. We discuss factors that lead to imprecision and inaccuracy; solutions for some of the major problems; resources and techniques to help standardize cholesterol measurement; and preanalytical issues that can affect cholesterol results—i.e., patient preparation; collection, processing, storage and proper analysis of the specimen; biological and seasonal variations; age and gender; diet; alcohol consumption; weight changes; exercise; primary diseases; and infections and trauma. Many of these can be controlled by the physician, resulting in more reliable cholesterol readings under stable metabolic conditions. Accurate values will help to classify the patient's coronary heart disease risk, define appropriate treatment strategies, and simplify monitoring of dietary and/or drug intervention.