Background: Artemether is a new and effective treatment for malaria, although relapse is a problem in monotherapy. These relapses could be related to a time-dependent decline in artemether plasma levels described in multiple-dose studies and probably caused by autoinduction. The aim of this study was to evaluate the effect of grapefruit juice on the decreasing bioavailability over time of artemether. Methods: In a randomized, two-phase crossover study, eight healthy male subjects took 100 mg oral artemether with 350 mL water or with 350 mL double-strength fresh frozen grapefruit juice once daily for 5 days. On day 1 and day 5, 17 blood samples were collected over a period of 8 hours. Results: The mean peak artemether plasma concentration (C(max)) and the mean area under the concentration-time curve (AUC) after the last dose at day 5 were about one third compared with day 1, without a change in the elimination half-life after intake with water (P = .006 for C(max); P = .005 for AUC) and with grapefruit juice (P < .001 for C(max) and AUC). Grapefruit juice increased C(max) (P = .021) and AUC (P < .001) twofold on day 1 (P = .021) and day 5 (P = .05 for C(max); P = .004 for AUC). Dihydroartemisinin, the active metabolite, showed a twofold increase in C(max) (P = .006) and AUG (P = .001) with grapefruit juice, without time- dependent changes of pharmacokinetic parameters. Conclusions: Grapefruit juice significantly increased the oral bioavailability of artemether but did not prevent the time-dependent reduction in bioavailability. It suggests that CYP3A4 in the gut wall is one of the metabolizing enzymes of artemether but seems to not be involved in the autoinduction process.