The incubation of human plasma very-low-density lipoprotein with human milk lipoprotein lipase results in an almost complete hydrolysis of triacylglycerols. The degradation of these substrates can be described by a consecutive reaction as follows: (Formula: see text), where k1, k2 and k3 are the apparent first-order rate constants of degradation. Using least-squares non-linear curve fitting, k1 and k2 are determined to be directly proportional to enzyme concentration. k1/k2 ratio of 1:12 is similar for both VLDL and trioleoylglycerol substrates of lipoprotein lipase. However, when trioleoylglycerol and rac-1,2-dioleoylglycerol are used as substrates, a direct measurement indicates a k1/k2 ratio of 1:1.5. This result suggests that the intermediary diacylglycerol produced by the lipoprotein reaction is incompletely re-equilibrated with the bulk of the substrate in the assay mixture. The k3 value is not proportional to lipoprotein lipase concentration, and in the enzyme concentration range studied, the value decreases when the enzyme concentration increases.