Melatonin, the pineal hormone, has now been widely used for various indications. Melatonin is principally metabolized by the cytochrome P450 system (CYP). To date, limited information regarding the effect of melatonin on hepatic drug metabolism has been provided. Therefore, this study was undertaken to examine the effect of melatonin on hepatic drug metabolism in Sprague-Dawley and Holtzman rats and in pigs. The in vitro studies in Sprague-Dawley rats and pigs clearly suggest that melatonin inhibited the activity of CYP1A1/2, 2B1, 2D1 and 3A2, whereas no inhibition was found on CYP2E1-catalytic activity. Melatonin showed a similar inhibitory profile on CYP-catalyzed metabolism in male and female Holtzman rats. Maximal inhibition was found with nonselective metabolism (e.g., 7-pentoxyresorufin O-depentylation, IC50 =104 muM) in rats. Melatonin preferentially inhibited CYP1A1 activity with IC50 values of 319 and 374 muM in pigs and rats, respectively. Subsequent studies examined the mechanism by which melatonin inhibited CYP activity and whether melatonin influenced the integrated phase I/phase II metabolism using liver slices from Sprague-Dawley rats. Melatonin inhibition was a competitive type toward CYP3A-catalyzed reactions in Sprague-Dawley rats and pigs. The findings from liver slices suggest that melatonin affects exclusively phase I metabolism. In vivo studies revealed that melatonin had no effect on total CYP content and NADPH-cytochrome c reductase activity, regardless of sex and strain of rats, dosage regimen or routes of administration (i.p. or oral). In microsomes from melatonin-treated rats, melatonin failed to inhibit the metabolism of most substrates, the exception being cortisol 6beta-hydroxylation catalyzed by CYP3A enzymes in male Holtzman rats. In humans, melatonin may interact with drugs catalyzed by CYP1A1/2 and 2B6 enzymes. Melatonin seems to be less likely to cause an interaction with drugs metabolized by the major isozymes responsible for drug metabolism in humans (e.g., CYP3A4, 2D6, 2C9). However, inhibition of some isozymes may be important, as implied by melatonin inhibition on CYP1A1/2 activity which may be involved in cancer prevention. Further studies using human samples and co-administration of melatonin with selective CYP-substrates (e.g., erythromycin) may provide more information on hepatic drug metabolism in humans.