The equilibria and kinetics of substitution of the 5 6-dimethylbenzimidazole at the alpha site of beta-(N-methylimidazolyl)cobalamin by N-methylimidazole have been investigated, and the product, bis(N-methylimidazolyl)cobalamin, has been characterized by visible and H-1 NMR spectroscopies. The equilibrium constant for (N-MeIm)Cbl(+) + N-MeIm reversible arrow (N-MeIm)(2)Cbl(+) was determined by H-1 NMR spectroscopy (9.6 +/- 0.1 M-1, 25.0 degreesC, I = 1.5 M (NaClO4)). The observed rate constant for this reaction exhibits an unusual inverse dependence on N-methylimidazole concentration, and it is proposed that substitution occurs via a base-off solvent-bound intermediate. Activation parameters typical for a dissociative ligand substitution mechanism are reported at two different N-MeIm(T) concentrations, 5.00 x 10(-3) M (DeltaH(double dagger) = 99 +/-2 kJ mol(-1), DeltaS(double dagger) = 39 +/- 5 J mol(-1) K-1, DeltaV(double dagger) = 15.0 +/- 0.7 cm(3) mol(-1), and 1.00 M (DeltaH(double dagger) = 109.4 +/- 0.8 kJ mol(-1), DeltaS(double dagger) = 70 +/- 3 J mol(-1) K-1, DeltaV(double dagger) = 16.8 +/- 1.1 cm(3) mol-'). According to the proposed mechanism,: these parameters correspond to the aquation of (N-MeIm)(2)Cbl(+) and the ring-opening reaction of the alpha -DMBI of (N-MeIm)Cbl(+) to give the solvent-bound intermediate in both cases, respectively.