The absorption of dietary cobalamin requires that a complex of intrinsic factor-cobalamin bind to specific receptors in the distal small intestine. The cellular location of these binding sites is unknown. To localize the intrinsic factor-cobalamin binding site, homogeneously purified hog intrinsic factor (7 pmol) and cobalamin (7 pmol) were instilled into ileal loops (n = 10) in anesthetized guinea pigs. Tissue controls for binding specificity were intrinsic factor-cobalamin in colon (n = 3) and jejunal (n = 3) loops, as well as intrinsic factor alone in ileal loops (n = 3). Intrinsic factor-[57Co]cobalamin was instilled in three ileal loops maintained for 90 min, and the radioactivity in the liver and kidneys was measured. Portions of each loop were washed in Krebs-Ringer buffer + Ca++, pH 7.4, or in either 1 mM Na2 ethylenediaminetetraacetic acid or in pH 4.5 acetate buffer. All tissue was processed for ultrastructural immunocytochemistry using a monospecific antisera to hog intrinsic factor as part of a well-characterized indirect immunoperoxidase method. Essentially all immunoreactive intrinsic factor-cobalamin was associated with the microvillous pits on mature ileal absorptive cells. Intrinsic factor binding was not present in colon or jejunum, or in the ileal loops instilled with intrinsic factor alone. Ethylenediaminetetraacetic acid and acid pH decreased intrinsic factor-cobalamin binding but caused severe disruption of the microvillous architecture. Absorption of [57Co]cobalamin was demonstrated, but intrinsic factor was not identified within enterocytes. The localization of specific intrinsic factor-cobalamin binding sites to the microvillous pit suggests that the intrinsic factor-cobalamin receptor may maintain a relatively fixed position within the surface membrane, and although alternative explanations are possible, our findings support the hypothesis that intrinsic factor is not internalized into the enterocyte during cobalamin absorption.