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The fractional esterification rate of cholesterol in apolipoprotein B (apoB)-depleted plasma (FERHDL) is a good indicator of particle size distribution in high density lipoprotein (HDL) and low density lipoprotein (LDL). However, there has been a discrepancy in the absolute values of FERHDL published by different laboratories. Because the main difference between the methods was in the labeling of lipoproteins with [3H]cholesterol we investigated the effect of using Corning immunoplates and paper discs as carriers of the labeled unesterified cholesterol. We found that Corning plates trap some 3H-labeled free cholesterol, which is released during incubation at 37°C. This means that this additional 3H-labeled free cholesterol is exposed to lecithin: cholesterol acyltransferase (LCAT) for a shorter time and artificially decreases FERHDL. Using paper discs discarded before incubation as carriers of the 3H-labeled free cholesterol results in complete labeling of HDL and thus yields higher values of FERHDL. —Dobiasova, M., L. Adler, T. Ohta, and J. Frohlich. Effect of labeling of plasma lipoproteins with [3H]cholesterol on values of esterification rate of cholesterol in apolipoprotein B-depleted plasma. J. Lipid Res. 2000. 41: 1356–1357.