Halofantrine (Hf) is a highly lipophilic antimalarial with poor and erratic absorption. Published data indicates that the oral bioavailability of Hf was increased 3-fold in humans and 12-fold in dogs when administered postprandially; however, the proportional formation of the active desbutyl metabolite (desbutylhalofantrine, Hfm) decreased 2.4-fold in humans and 6.8-fold in dogs (Milton et al., Br. J. Clin. Pharmacol. 1989, 28, 71-77; Humberstone et al., J. Pharm. Sci. 1996, 85, 525-529). The current study was undertaken to confirm the putative involvement of CYP3A4 in the N-dealkylation of Hf to Hfm by administering Hf with and without ketoconazole (KC), a specific CYP3A4 inhibitor, and measuring the resulting plasma concentration profiles of Hf and Hfm. The plasma Hfm/Hf AUC(0-72 h) ratio after fasted oral administration of Hf without KC was 0.56, whereas the ratio after fasted oral administration with KC was less than 0.05. It is likely that both hepatic and prehepatic (enterocyte-based) CYP3A4 contributed to metabolism of Hf to Hfm after oral administration. Interestingly, the low plasma Hfm/Hf AUC ratios observed after fasted administration of Hf with KC were similar to the low values previously observed when Hf was administered postprandially (despite increased Hf absorption). The mechanism(s) by which postprandial administration of Hf led to a decrease in its metabolism are unknown, but based on the current data, could include inhibition of CYP3A4-mediated metabolism by components of the ingested meal. Other possibilities include a lipid-induced postprandial recruitment of intestinal lymphatic transport or avoidance of metabolism during transport through the enterocyte into the portal blood. Further studies are required to determine the relative contributions by which these different processes may decrease the presystemic metabolism of Hf.