The effects of freezing and storage at − 20° C of serum on the measurement of cholesterol and triglycerides concentrations in lipoprotein fractions separated by density gradient ultracentrifugation were investigated. Fresh, fasting, normolipidemic sera from 24 healthy individuals were divided into aliquots and stored, using different conditions. After storage, serum lipoproteins were separated by density gradient ultracentrifugation, and cholesterol and triglycerides assayed enzymatically. Freezing as such had no effect on the concentration of cholesterol and triglycerides in the various lipoproteins; changes averaged 0.00 mmol/l (range, - 0.02 to 0.02 mmol/l). The concentration of cholesterol in the major cholesterol fractions (LDL, HDL2 and HDL3) changed on average by < 4.1% over the full period of 27 wk. The percentage changes in the triglyceride concentrations over the first 11 wk were < 5% for the major triglyceride fractions (VLDL and LDL) except for LDL after 11 weeks of storage (change 9.4%). By 27 weeks of storage, triglyceride concentrations had changed by -13.0% in VLDL and by 13.0% in LDL. It is concluded that fasting normolipidemic serum can be stored at −20°C for at least 11 wk before lipoproteins are separated by density gradient ultracentrifugation and analyzed for cholesterol and triglycerides.