We have studied the interaction between transcobalamin (TC) and the ligands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Unsaturated TC was adsorbed on CM-Sepharose in order to ensure fast separation of the matrix-bound protein from the reaction medium. The forward reaction TC+Cbl-->TC-Cbl (rate constant k+Cbl) and the backward reaction TC-Cbl-->TC+Cbl (k-Cbl) were followed in time. A single-step binding model (with no intermediate protein-ligand complex) was sufficient to fit the data. The calculated rate constants were k+Cbl = 0.6 nM-1 min-1 and k-Cbl = 1.3 x 10(-4) min-1, which corresponded to the TC-Cbl dissociation constant KDCbl = 0.2 pM. Reaction between TC and Cbl developed against electrostatic forces, and the effective charges of the interacting species were estimated as both +1 or both -1. The competition between Cbl and Cbi for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k+Cbi = 0.03 nM-1 min-1, k-Cbi = 0.03 min-1, and KDCbi = 1 nM. Slow dissociation of TC-Cbl guarantees its stability in plasma for 5-10 h, while Cbi bound to TC would be transferred to haptocorrin in less than 1 h.