This study reports the development of a specific and sensitive liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) assay for the quantification of the in vitro O-glucuronidation of chloramphenicol (CP), the determination of the kinetic parameters for the O-glucuronidation of CP in pooled human liver microsomes (HLM), the biosynthesis of the CP glucuronides (CPGlu), and identification of the structures of CPGlu by (1)H-nuclear magnetic resonance (NMR) and MS. Two glucuronyl derived metabolites of CP were obtained from the incubation of alamethicin-activated HLM with CP and uridine 5'-diphosphoglucuronic acid (UDPGA) in pH 7.4 TRIS buffer. Their identification and structural confirmation were achieved by beta-glucuronidase hydrolysis, in the presence and absence of UDPGA, and by (1)H-NMR and LC-MS/MS. These two metabolites were biosynthesized, isolated, and purified using high-performance liquid chromatography (HPLC). Their structures were further identified as the 1-O-CPGlu (the minor glucuronide formed at the secondary alcohol of CP) and 3-O-CPGlu (the major glucuronide formed at the primary alcohol of CP) by LC-MS/MS and two-dimensional NMR. The enzymatic kinetic parameters K(m) and V(max) in HLM for the 3-O-CPGlu were determined to be 650 microM and 0.26 nmoles min(-1) mg(-1), respectively, and for the 1-O-CPGlu to be 301 microM and 0.014 nmoles min(-1) mg(-1), respectively. This study also provides a sensitive and specific method for the measurement of in vitro CP-UDP-glucuronosyltransferase (UGT) activity.