The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 microM with human liver microsomes (4 mg ml(-1)) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.