Carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are the major hydrolytic enzymes responsible for the metabolism of numerous therapeutic agents as well as endogenous substrates. CES1 and CES2 differ distinctly in their substrate specificity and tissue distribution. In this study, we investigated the role of CES1 and CES2 in converting the antihypertensive prodrug trandolapril to its more active form trandolaprilat, and determined the influence of two newly identified CES1 mutations p.Gly143Glu and p.Asp260fs on trandolapril metabolism. Western blot analysis demonstrated that CES1 is expressed in human liver microsomes (HLM) but not in human intestinal microsomes (HIM). In vitro incubation studies were conducted to contrast the enzymatic activity of HLM as well as HIM upon trandolapril hydrolysis. Trandolapril was rapidly hydrolyzed to its principal active metabolite trandolaprilat after incubation with HLM. In contrast, in HIM, where CES2 is predominantly expressed, incubations did not produce any detectable trandolapril hydrolysis. Furthermore, hydrolysis of trandolapril catalyzed by wild type (WT) and mutant CES1 were assessed utilizing transfected Flp-In-293 cells stably expressing WT CES1 and two variants. WT CES1 efficiently hydrolyzed trandolapril to trandolaprilat with V(max) and K(m) values of 103.6+/-2.2 nmole/min/mg protein and 639.9+/-32.9muM, respectively. However, no appreciable trandolapril hydrolysis could be found after incubation with both p.Gly143Glu and p.Asp260fs variants. Thus, trandolapril appears to be a CES1 selective substrate while CES2 exerts little to no catalytic activity towards this compound. CES1 mutations p.Gly143Glu and p.Asp260fs are essentially dysfunctional enzymes with regard to the conversion of trandolapril to its more active metabolite trandolaprilat.