Hamster intrinsic factor (IF) preparations markedly enhanced the uptake of (57)cobalt-labeled cyanocobalamin (B(12)-(57)Co) by brush borders and microvillous membranes isolated from villous absorptive cells obtained from the distal but not the proximal half of hamster intestine. A similar effect was observed with rat and rabbit IF preparations, but IF preparations obtained from man, dog, and hog were ineffective. After fractionation of hamster IF preparations by gel filtration or ion exchange chromatography, the extent to which each fraction enhanced B(12)-(57)Co uptake by brush borders correlated closely with the vitamin B(12) binding capacity of the fraction. IF-mediated attachment of B(12)-(57)Co to brush borders occurred rapidly, was not diminished by removal of glucose or oxygen from the incubation medium, and was not significantly altered when incubation temperatures were reduced from 37 degrees C to 7 degrees C. Marked reduction in uptake occurred, however, in the absence of divalent cations. IF enhanced B(12)-(57)Co uptake by brush borders isolated from the proximal half of the intestine when these proximal brush borders were preincubated with supernatant fluid obtained after centrifugation of homogenates of distal intestinal mucosa at 28,500 g. The factor in this supernate responsible for the effect on proximal brush borders was shown to be particulate in nature upon centrifugation at speeds of 54,500 g or greater. The resultant pellet contained ribosomes and membranous fragments. Prolonged incubation of brush borders with crude saline extracts of hamster gastric mucosa resulted in decreased uptake of B(12)-(57)Co and marked lysis of brush borders with concomitant release of tissue nitrogen. Neither lysis of brush borders nor decreased uptake of B(12)-(57)Co with prolonged incubation was observed when hamster IF was partially purified. Furthermore, uptake of B(12)-(57)Co by brush borders increased with increasing purity of the IF preparation used. These results demonstrate IF-mediated attachment of B(12)-(57)Co to brush borders and microvillous membranes of hamster intestinal cells and provide further support for the presence of a specific receptor for IF-bound vitamin B(12) at the microvillous surface of the intestinal cell. IF-mediated attachment to the intestinal cell surface appears to be facilitated by divalent cations and to result from adsorption rather than an energy-requiring enzymatic reaction. Crude sources of hamster IF contain a factor which causes lysis of brush borders in vitro and which may explain in part the inhibitory effects of IF excess previously observed in vitro.