This study assessed the mechanism of uptake of biotin by the fetal-facing (basolateral) membrane of the term human placenta. Using membrane vesicles, we showed that most of the uptake was attributable to transfer of the vitamin into the vesicle and that the uptake was saturable, Na-dependent, carrier-mediated, and electroneutral. The rate of uptake was less than for biotin uptake by the maternal-facing (apical) membrane of the human placenta. Because ethanol inhibits biotin uptake by the apical membrane, the effect of ethanol on uptake by basolateral vesicles was investigated. With 10-hr exposure at a concentration of 2 and 3 mg/ml, but not 1 mg/ml, ethanol modestly inhibited biotin uptake. The mechanism of inhibition by alcohol is not known.