Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed angiotensin-converting enzyme inhibitors: enalapril and ramipril. Here, we studied recombinant human CES1- and CES2-mediated hydrolytic activation of the prodrug esters enalapril and ramipril, compared with the activation of the known substrate trandolapril. Enalapril, ramipril, and trandolapril were readily hydrolyzed by CES1, but not by CES2. Ramipril and trandolapril exhibited Michaelis-Menten kinetics, while enalapril demonstrated substrate inhibition kinetics. Intrinsic clearances were 1.061, 0.360, and 0.02 ml/min/mg protein for ramipril, trandolapril, and enalapril, respectively. Additionally, we screened a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug interactions. The findings in the present study may contribute to the prediction of such interactions in humans, thus opening up possibilities for safer drug treatments.