The antiestrogen tamoxifen (Tam or Nolvadex, ICI)-Z-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1,2-diphenyl-1-butene is widely used in treatment of hormone-dependent breast cancer. The drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-desmethyl metabolite (DMT). This study has been carried out to determine the P450 enzyme involved in the N-oxidative demethylation of Tam in microsomal samples from 25 human livers (23 adults, two children). This metabolic step was inhibited by carbon monoxide up to 75%. Tam was demethylated into DMT with an apparent Km of 98 +/- 10 microM; rates varied between 37 and 446 pmol/min/mg microsomal protein. These metabolic rates were strongly correlated with 6 beta-hydroxylation of testosterone (r = 0.83) and erythromycin N-demethylase (r = 0.75), both activities known to be associated with P450 IIIA enzyme. To further assess whether or not the Tam demethylation pathway is catalysed by the same P450, the inhibitory effect of TST on this reaction was determined. The competitive inhibition had an apparent Ki of 100 +/- 10 microM. Drugs such as erythromycin, cyclosporin, nifedipine and diltiazem were shown to inhibit in vitro the metabolism of tamoxifen. Furthermore the P450 IIIA content of liver microsomal samples, measured by Western blot technique using a monoclonal P450NF (nifedipine) antibody, was strongly correlated with DMT formation (r = 0.87). Tam N-demethylase activity was inhibited by more than 65% with polyclonal anti-human anti-P450NF. All these in vitro observations establish that a P450 enzyme of the IIIA sub-family is involved in the oxidative demethylation of tamoxifen in human liver.