At a concentration of 1 x 10–5 m, biotin-H3 and biotin-C14 were transported against a concentration gradient by hamster everted intestinal sacs. Similar transport was shown by the small intestine of the white mouse, Mongolian gerbil, chinchilla, flying squirrel, round-tail squirrel, Malayan black squirrel, and chipmunk (and was maximal in the chipmunk intestine). Transport under these conditions was not shown by the small intestine of the rat, rabbit, guinea pig, ferret, or carp. There does not appear to be a correlation between the need for biotin as a vitamin in a species and the presence of this transport system. Chromatography of the mucosal and serosal solutions did not reveal any radioactive spot other than the original biotin-H3 or biotin-C14. This evidence, plus the inhibition of transport by avidin, suggests that it is biotin that is being transported, and not a metabolite. The unidirectional flux of biotin (from a side containing the vitamin to a side which did not), was uninfluenced by the addition of avidin to the buffer into which diffusion was occurring. The "apparent Km" for biotin transport was on the order of 6 x 10–5 m, based on equilibrium measurements. Transport of biotin in the hamster small intestine occurred principally in the first one-third to one-half of the small intestine. Amino acids, urea, biotin-d-sulfoxide, as well as biotin-l-sulfoxide did not inhibit biotin transport. Biocytin, biotin methyl ester, diaminobiotin, desthiobiotin, thioctic acid, and two other analogues inhibited biotin transport.