Transport of biotin was examined in rat intestine using the everted sac technique. Transport of 0.1 microM biotin was linear with time for at least 30 min of incubation and occurred at a rate of 3.7 pmol X g initial tissue wet wt-1 X min-1. Transport of biotin was higher in the jejunum than the ileum and was minimum in the colon (85 +/- 6, 36 +/- 6, and 2.8 +/- 0.6 pmol X g initial tissue wet wt-1 X 25 min-1, respectively). In the jejunum, transport of biotin was saturable at low concentrations (Kt = 3.73, microM, Vmax = 3.11 nmol X g initial tissue wet wt-1 X 25 min-1) but linear at higher concentrations (greater than 10 microM). The transport of low concentrations of biotin was inhibited by structural analogues (desthiobiotin, biotin methyl ester, diaminobiotin, and biocytin), Na+ dependent, energy dependent, temperature dependent, and proceeded against a concentration gradient in the serosal compartment. No metabolic alteration occurs to the biotin molecule during transport. This study demonstrates that biotin transport in rat intestine occurs by a carrier-mediated process at low concentrations and by simple diffusion at high concentrations. Furthermore, the carrier-mediated process is Na+, energy, and temperature dependent.