The antiviral drug zidovudine (ZDV) and the analgesic and antipyretic agent acetaminophen (AP) are both biotransformed in the liver of humans to ether glucuronides. A previous clinical trial of ZDV suggested the potential for a clinically significant interaction between AP and ZDV, probably based upon competing hepatic metabolism. To study the mechanism of this potential competition between AP and ZDV as substrates for uridine diphosphoglucuronyltransferase (UDPGT), enzyme kinetic studies were performed using rat liver microsome preparations. Cross inhibition studies demonstrated that AP glucuronidation was competitively inhibited by ZDV, while ZDV glucuronidation was slightly inhibited by AP in a noncompetitive interaction.