The apical (AP)-to-basolateral (BL) transport of [3H]biotin across Caco-2 cell monolayers grown on a Transwell polycarbonate membrane was shown to be dependent on days in culture, reaching a maximum at 10 days. The AP-to-BL transport of [3H]biotin was always much greater than the BL-to-AP transport. AP-to-BL transport of [3H]biotin was concentration-(Km = 1.49 microM and Vmax = 1.86 pmol.mg protein-1.minute-1), temperature-, Na(+)- and energy-dependent, and proceeded against a concentration gradient in the BL side. [3H]Biotin transport was inhibited by unlabeled biotin and several structural analogues (e.g., desthiobiotin and biotin methyl ester) but not by taurocholic acid. No metabolism of [3H]biotin was detected in the AP-to-BL transport across Caco-2 cell monolayers. This study demonstrates that [3H]biotin transport in Caco-2 cells occurs by a carrier-mediated process.