The biliary excretion of the major methadone metabolites was studied after the administration of purified 14C-labeled metabolites of methadone. The three methadone metabolites administered to rats were the pyrrolidine and pyrroline metabolites and the glucuronide conjugate of the hydroxylated pyrroline metabolite. We found that the excretion of 14C into bile was greater after administration of the metabolites which underwent fewer metabolic steps prior to biliary excretion. Phenobarbital pretreatment of rats increased the biliary excretion of 14C after the administration of the 14C-labeled pyrrolidine and pyrroline metabolites, both of which undergo further metabolism. Phenobarbital pretreatment did not change the biliary excretion of 14C-labeled glucuronide of the pyrroline metabolite which does not undergo further metabolism in the liver. Analysis of bile samples showed that the increased biliary excretion of 14C in phenobarbital-pretreated rats after administration of the labeled pyrrolidine and pyrroline metabolites was due primarily to increases in the biliary excretion of methadone metabolites which resulted from further in vivo metabolism of the injected metabolites. These data confirm our earlier suggestion (Roerig et al., Biochem. Pharmacol. 24: 355-362, 1975) that phenobarbital pretreatment increases biliary excretion of methadone metabolites by increasing hepatic metabolism of methadone. Furthermore, it can be concluded from these studies that metabolism is a rate-limiting process in the excretion of methadone metabolites in bile.