A receptor-binding region of intrinsic factor (IF) has been localized to amino acid residues 25-44 by in vitro transcription/translation studies. To further define sites within the region that are necessary for binding, the effects on binding activity of brush border membranes were tested when peptides corresponding with residues 1-44 were added to normal IF and when point mutations were made between residues 25 and 44. Both human IF and IF-cobalamin (cbl) complex bound equally to membranes. None of the peptides tested inhibited human IF or IF-cbl complex binding. Both control rat IF and rat IF that was mutated to be like human IF in residues 25-44 bound to guinea pig and rat membranes; the mutant with altered charge showed 50- to 100-fold decreased affinity. Thus, the putative receptor binding region is important, but cannot alone account for all the physiologic parameters of IF binding. Conformational changes in IF are additionally important.