Following oral administration of [14C]-gefitinib to albino and pigmented rats, radioactivity was widely and rapidly distributed, with the highest levels being found in liver, kidney, lung and gastrointestinal tract, but with only low levels penetrating the brain. Levels of radioactivity persisted in melanin-containing tissues (pigmented eye and skin). Binding to plasma proteins was high (86-94%) across the range of species examined and was 91% in human plasma. Substantial binding occurred to both human serum albumin and alpha-1 acid glycoprotein. Following oral and intravenous administration of [14C]-gefitinib, excretion of radioactivity by rat, dog and human occurred predominantly via the bile into faeces, with < 7% of the dose being eliminated in urine. In all three species, gefitinib was cleared primarily by metabolism. In rat, morpholine ring oxidation was the major route of metabolism, leading to the formation of M537194 and M608236 as the main biliary metabolites. Morpholine ring oxidation, together with production of M523595 by O-demethylation of the quinazoline moiety, were the predominant pathways in dog, with oxidative defluorination also occurring to a lesser degree. Pathways in healthy human volunteers were similar to dog, with O-demethylation and morpholine ring oxidation representing the major routes of metabolism.