The effect of the anticonvulsant drugs carbamazepine and primidone on the transport of biotin in the human intestine was examined with purified brush border membrane vesicles (BBMVs) and basolateral membrane vesicles (BLMVs). Both agents inhibited biotin transport in BBMV in a concentration-dependent manner. The inhibition by both carbamazepine and primidone was competitive (inhibition constant [Ki] of 4.70 and 2.25 mmol/L, respectively) and appeared to be specific because the transport of D-glucose was not affected by different concentrations of these pharmacologic agents. The transport of biotin in BLMV was not affected by carbamazepine or primidone. These results demonstrate that carbamazepine and primidone are competitive inhibitors of biotin transport in the human intestine and that the inhibitory effect is directed toward the substrate transport system at the brush border membrane of the enterocyte. These findings may relate to possible impairment of biotin status in patients on long-term therapy with anticonvulsant agents.