Clinical observation of a synergistic effect of ketamine on morphine analgesia remains controversial. Although a pharmacodynamic basis for an interaction has been explored in animal and clinical studies, the possibility of a pharmacokinetic mechanism has not been investigated. Whereas both morphine and morphine-6-glucuronide are effective analgesics, morphine-3-glucuronide (M3G) lacks activity. Thus, changes in the metabolism and disposition of morphine may result in an altered response. First, we investigated the interaction between morphine and ketamine in the isolated perfused rat liver preparation. The clearance of morphine was decreased from 16.8 ± 4.6 ml/min in the control period to 7.7 ± 2.8 ml/min in the ketamine-treatment period, with the formation clearance of M3G decreasing from 8.0 ± 4.1 ml/min to 2.1 ± 1.1 ml/min. Fractional conversion of morphine to M3G was significantly decreased from 0.46 ± 0.17 in the control period to 0.28 ± 0.14 upon the addition of ketamine. The possible mechanism of the interaction was further investigated in vitro with rat liver microsomes as the enzyme source. The formation of M3G followed single-enzyme Michaelis-Menten kinetics, with a mean apparent Km of 2.18 ± 0.45 mM and Vmax of 8.67 ± 0.59 nmol/min/mg. Ketamine inhibited morphine 3-glucuronidation noncompetitively, with a mean Ki value of 33.3 ± 7.9 μM. The results demonstrate that ketamine inhibits the glucuronidation of morphine in a rat model.