The rates of demethylation and hydroxylation of amitriptyline, nortriptyline, and 10-hydroxyamitriptyline by microsomes from adult human livers were determined by use of mass-fragmentographic or liquid-chromatographic quantitation of the formed metabolites. The demethylation rates of amitriptyline and 10-hydroxyamitriptyline were higher than the hydroxylation rates of amitriptyline and nortriptyline, especially at high substrate concentration. The amitriptyline demethylation rates were 96-570 and 1750-9230 pmol per mg of protein per 10 min at substrate concentrations of 5 and 100 micro M, respectively. The corresponding rates for the hydroxylations were 43-146 and 305-871, respectively. At high substrate concentration (250 micro M) the curve of concentration vs. rate for 10-hydroxylation of amitriptyline seemed to approach a plateau, whereas those for demethylation did not. Interaction between amitriptyline and nortriptyline at the microsomal level was studied by use of deuterium-labeled amitriptyline, and these two compounds were found to inhibit the hydroxylation of each other. In contrast to hydroxylation, the demethylation of labeled amitriptyline increased upon addition of nortriptyline. These results suggest that the well-established variation in steady-state plasma levels of tricyclic antidepressants is due to interindividual differences in liver enzyme activity.