The incubation of 1-methyltetrahydro-beta-carboline (1-MeTHBC) with hepatocytes isolated from 3-methylcholanthrene-treated rats led to formation of the 5-, 6- and 7-hydroxylated products. The hydroxylating activity was associated with the microsomal fraction as indicated by testing different subcellular fractions. The highest activity for hydroxylating 1-MeTHBC was found in liver which was about ten times as active as lung. Only a trace amount of hydroxylating activity was present in brain and kidney tissue. Analysis using chiral gas chromatography revealed an unequal abundance of enantiomers in all three products. The formation of the 5-, 6- and 7-hydroxylated products was confirmed in vivo by analysis of 24 h urine samples after intraperitoneal administration of 1-MeTHBC to 3-methylcholanthrene-treated rats.