Although there is evidence for an important role of UGT2B10 in the N-glucuronidation of drugs and other xenobiotics, the inhibitor selectivity of this enzyme is poorly understood. This study sought primarily to characterize the inhibition selectivity of UGT2B10 by UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors used for reaction phenotyping, and 34 antidepressant and antipsychotic drugs that contain an amine functional group. Initial studies demonstrated that cotinine is a highly selective substrate of human liver microsomal UGT2B10. The kinetics of cotinine N-glucuronidation by recombinant UGT and human liver microsomes (± bovine serum albumin) were consistent with the involvement of a single enzyme. Of the UGT enzyme-selective inhibitors employed for reaction phenotyping, only the UGT2B4/7 inhibitor fluconazole reduced recombinant UGT2B10 activity to an appreciable extent. The majority of antidepressant and antipsychotic drugs screened for effects on UGT2B10 inhibited enzyme activity with IC50 values <100 mM. The most potent inhibition was observed with the tricyclic antidepressants amitriptyline and doxepin and the tetracyclic antidepressant mianserin, and the structurally related compounds desloratadine and loratadine. Molecular modeling using a ligand-based approach indicated that hydrophobic and charge interactions are involved in inhibitor binding, whereas spatial features influence the potency of UGT2B10 inhibition. Respective mean Ki,u (± S.D.) values for amitriptyline, doxepin, and mianserin inhibition of human liver microsomal UGT2B10 were 0.61 ± 0.05, 0.95 ± 0.18, and 0.43 ± 0.01 mM. In vitro-in vivo extrapolation indicates that these drugs may perpetrate inhibitory drug-drug interactions when coadministered with compounds that are cleared predominantly by UGT2B10.