We developed a simple method for the quantitation of triglycerides in electrophoretically separated lipoproteins by specific enzymatic staining. After electrophoresis, glycerol is liberated from triglycerides by the action of cholesterol esterase. Glycerol is oxidized by a sequence of enzymatic reactions. Due to the presence of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in the reaction mixture, two moles of the precipitating dye formazane are generated per mole glycerol. The relative amounts of alpha, pre-beta, and beta lipoproteins are determined by densitometric scanning at 570 nm. Absolute triglyceride concentrations of the respective lipoprotein fractions are calculated from total triglycerides. When tested with purified very low density lipoproteins, the electrophoresis assay was linear between 0.08 and 6.5 g/l pre-beta lipoprotein triglycerides. The intra-assay and inter-assay coefficients of variation were between 5.2% and 9.8%, and between 3.2% and 12.9%, respectively. Comparison of the electrophoresis method with a combined ultracentrifugation/precipitation method in 172 sera resulted in the following correlation coefficients: alpha lipoprotein versus high density lipoprotein triglycerides, r = 0.847; pre-beta lipoprotein versus very low density lipoprotein triglycerides, r = 0.989; beta lipoprotein versus low density lipoprotein triglycerides, r = 0.815. This method is easy to perform, and is a precise and accurate technique for the determination of lipoprotein triglycerides. It is the first reliable method that allows the direct quantification of LDL triglycerides without ultracentrifugation.