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 beta-naphthol were synthesized and characterized as substrates for rabbit (rCE) and human liver (hCE1) carboxylesterases. All of the esters of o-, p-nitrophenols, and beta-naphthols showed moderate hydrolysis by both rCE and hCE1. Esters of beta-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 V(max) of 54.4 micromoles/min/mg, and a K(m) value of 1071 microM. 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.