A major metabolic pathway of haloperidol is glucuronidation catalyzed by UDP-glucuronosyltransferase (UGT). In this study, we found that two glucuronides were formed by the incubation of haloperidol with human liver microsomes (HLM) and presumed that the major and minor metabolites (>10-fold difference) were O- and N-glucuronide, respectively. The haloperidol N-glucuronidation was catalyzed solely by UGT1A4, whereas the haloperidol O-glucuronidation was catalyzed by UGT1A4, UGT1A9, and UGT2B7. The kinetics of the haloperidol O-glucuronidation in HLM was monophasic with K(m) and V(max) values of 85 μM and 3.2 nmol · min⁻¹ · mg⁻¹, respectively. From the kinetic parameters of the recombinant UGT1A4 (K(m) = 64 μM, V(max) = 0.6 nmol · min⁻¹ · mg⁻¹), UGT1A9 (K(m) = 174 μM, V(max) = 2.3 nmol · min⁻¹ · mg⁻¹), and UGT2B7 (K(m) = 45 μM, V(max) = 1.0 nmol · min⁻¹ · mg⁻¹), we could not estimate which isoform largely contributes to the reaction. Because the haloperidol O-glucuronidation in a panel of 17 HLM was significantly correlated (r = 0.732, p < 0.01) with zidovudine O-glucuronidation, a probe activity of UGT2B7, and the activity in the pooled HLM was prominently inhibited (58% of control) by gemfibrozil, an inhibitor of UGT2B7, we surmised that the reaction would mainly be catalyzed by UGT2B7. We could successfully estimate, using the concept of the relative activity factor, that the contributions of UGT1A4, UGT1A9, and UGT2B7 in HLM were approximately 10, 20, and 70%, respectively. The present study provides new insight into haloperidol glucuronidation, concerning the causes of interindividual differences in the efficacy and adverse reactions or drug-drug interactions.