The uptake of biotin and biocytin was investigated in rat intestine using the everted sac technique. It has been shown that at biotin and biocytin concentrations less than 40 and 50 nM respectively, absorption proceeds by a saturable process, whereas at higher concentrations uptake by passive diffusion predominates. Fractionation of solubilized brush border preparations indicates that biotinidase is the only protein which binds biotin in this preparation.