The purpose was to study in vitro metabolism of 2',3',5'-triacetyl-6-azauridine (1) by porcine liver esterase (PLE) and in human plasma using an automated analytical system developed previously. A gradient-LC method was developed to study the concentration-time course of 1 and its metabolites. A fast-LC assay was used to study the temperature effect on the metabolism of 1 by the PLE. 1 and all of its proposed possible metabolites were separated by the gradient-LC method in less than 10 min. Two simplified kinetic schemes were developed to describe the time course of 1, the intermediates and final metabolites with only five rate constants for the metabolisms of 1 by PLE and four rate constants in human plasma. Both enthalpy and entropy of activation in the in vitro metabolism of 1 by PLE were obtained.