In an effort to develop novel prodrugs for viral directed enzyme prodrug therapy (VDEPT) approaches to chemotherapy, eleven esters and carbamates of o-nitrophenol, p-nitrophenol, and β-naphthol were synthesized and characterized as substrates for rabbit (rCE) and human liver (hCE1) carboxylesterases. All of the esters of o-, p-nitrophenols, and β-naphthols showed moderate hydrolysis by both rCE and hCE1. Esters of β-naphthols exhibited higher hydrolysis rates compared to esters of p-nitrophenols by rCE. Of the carbamates, 4-benzyl-piperazine-1-carboxylic acid 2-nitrophenol showed preferential hydrolysis by rCE compared to hCE1 with a Vmax of 54.4 μmoles/min/mg, and a Km value of 1071 μM. Substrate metabolism by a specific CE or inhibition of CEs by each compound depended on several factors, including the types of functional groups and linking moieties. © 2003 Elsevier Science Ltd. All rights reserved.