The influence of phenobarbital (PB) on the hepatobiliary disposition of acetaminophen (APAP), acetaminophen glucuronide (AG) and acetaminophen sulfate (AS) was examined in the recirculating isolated perfused rat liver (IPL). PB was administered as a 5-mumol bolus to the IPL (acute) or in vivo (75 mg/kg/day, i.p., x 5 days), followed by a 48-hr washout, before addition of an APAP (66-mumol) bolus to the IPL. Acute PB administration to the IPL did not affect the total hepatic clearance of APAP or the formation clearance to AG and AS. The rate constant for the biliary excretion of AG and the percentage of the dose recovered in bile as AG were decreased significantly after the acute PB dose. In vivo PB pretreatment induced significantly the UDP-glucuronyltransferases (i.e., an increase in APAP clearance, the formation clearance to AG and in the estimate of the AG formation rate constant). The estimate of the AG sinusoidal egress rate constant after in vivo PB pretreatment also was increased significantly. The AG biliary excretion rate constant and the percentage of the dose recovered in bile as AG were decreased approximately 5-fold after in vivo PB pretreatment, similar to values obtained after acute PB administration. PB was detected only after acute PB administration, whereas unconjugated p-hydroxyphenobarbital was not detected after either treatment. These data indicate that neither PB itself nor an induction effect related to in vivo PB pretreatment are required directly to impair hepatobiliary disposition.