This study was conducted to investigate the effect of rifampin on the pharmacokinetics of bosentan. Healthy male subjects received bosentan 125 mg b.i.d. for 6.5 days in the presence or absence of rifampin 600 mg once a day. In vitro experiments were performed to investigate the effect of rifampin on the uptake of bosentan into Chinese hamster ovary cells expressing the human organic anion-transporting polypeptide (OATP)1B1, -1B3, and -2B1. Following the first concomitant administration, there was a fivefold increase in bosentan trough concentrations. At steady state, concomitant rifampin significantly decreased exposure to bosentan by 58%. Rifampin potently inhibited the uptake of bosentan into cells expressing human OATP1B1 and -1B3. Rifampin decreased the exposure to bosentan consistent with its known cytochrome P450 enzyme-inductive properties. The initial increase in bosentan concentrations can be explained by an inhibitory effect of rifampin on hepatic drug transporters.