Five milligram doses of d-, l-, or dl-methadone [an oral narcotic analgesic] were added to the perfusate of the isolated perfused rat liver and the disposition of each compound was monitored over a 120 min period. There was an initial rapid decline in concentration of each drug in the perfusate, with approximately 70% disappearing in the first 10 min of the perfusion. Thereafter, the rate of decline of each compound slowed and the apparent half-life was about 100 min. In experiments with d-, l- and dl-methadone the mean hepatic extraction from the perfusate at 10 min was 72, 66 and 69%, respectively. A comparison of the rate of disappearance of the d- and the l-isomers from the perfusate showed that the concentration of the d-isomer at each sampling time was lower than the concentration of the l-isomer and this concentration ratio ranged between 68 and 79%. A study of the disposition of dl-methadone in the isolated perfused liver indicated that the initial rapid decline of the drug in the perfusate in the first 10 min was caused primarily by the uptake of the unchanged drug into the liver. Thereafter, the methadone concentration in the perfusate and liver declined slowly with time and was paralleled by an increase in the concentration of the primary mono-N-demethylated metabolite, which appeared in the liver, perfusate and bile. Smaller quantities of more polar metabolites, including hydroxylated compounds and conjugates, were also present in the liver and bile. Addition of SKF 525-A [2-diethylaminoacethyl 2,2-diphenylvalerate hydrochloride] retarded the rate of methadone decline from the perfusate and pretreatment of the rat with phenobarbital increased the rate of biotransformation of methadone in the isolated perfused liver.