Bisubstrate reaction kinetics and product inhibition studies were used to characterize the kinetic mechanism of a partially purified uridine diphosphate glucuronosyltransferase (UDPGT). These studies indicate that the reaction most likely occurs via a random order sequential mechanism. The effect of electron withdrawing and donating groups on the rate of reaction was also determined. It was found that electron donating groups increased the rate of glucuronide conjugation. This result is consistent with nucleophilic attack of the C-1 carbon of the UDP-glucuronic acid (UDPGA) by an SN2 mechanism. This is the first direct evidence for a SN2 mechanism in UDPGT catalysis.