Three proteins, intrinsic factor (IF), transcobalamin (TC), and haptocorrin (HC), all have an extremely high affinity for the cobalamins (Cbls, Kd approximately 5 fM) but discriminate these physiological ligands from Cbl analogues with different efficiencies decreasing in the following order: IF > TC > HC. We investigated interactions of these proteins with a number of ligands: Cbl, fluorescent conjugate CBC, two base-off analogues [pseudo-coenzyme B12 (pB) and adenosyl factor A (fA)], and a baseless corrinoid cobinamide. Protein-ligand encounter and the following internal rearrangements in both molecules were registered as a change in the fluorescence of CBC (alone or mixed with other ligands), a transition in absorbance of pB and fA (base-off --> on-base conversion), and alterations in the molecular mass of two split IF domains. The greater complexity of the binding kinetics followed better Cbl specificity (HC < TC < IF). On the basis of the experimental results, we propose a general binding model with three major steps: (1) initial attachment of the ligand to the high-affinity C-domain, (2) primary assembly of N- and C-domains, and (3) slow adjustments and fixation of the ligand at the domain-domain interface. Since step 3 was characteristic of highly specific TC and especially IF, we suggest its particular importance for ligand recognition. The designed models revealed the absolute Kd values for a group of analogues. Calculations show that most of them could potentially bind to the specific transporters IF and TC under physiological conditions. Implications of this finding and the protective role of HC are discussed.