We describe an enzymatic method for rapid, precise measurement of serum triglycerides with use of sample:reagent ratios as large as 1:200. Hydrolysis of triglycerides is catalyzed by lipase to produce glycerol and free fatty acids. The glycerol generated is then phosphorylated by adenosine 5'-triphosphate in the presence of glycerol kinase. Oxidation of the resulting glycerol 3-phosphate to produce hydrogen peroxide is catalyzed by L-alpha-glycerophosphate oxidase. An intense red chromogen is produced by the peroxidase-catalyzed coupling of 4-aminoantipyrene and sodium 2-hydroxy-3,5-dichlorobenzenesulfonate with hydrogen peroxide. This sensitive chromogen system not only permits use of unusually small sample volumes, it also facilitates a linear response to serum triglyceride concentrations up to at least 10 g/L while displaying good Ringbom (measure of accuracy) characteristics.