The reduced form of aquacobalamin binds nitric oxide very effectively to yield a nitrosyl adduct, Cbl(II)-NO. UV-vis, H-1-, P-31-, and N-15 NMR data suggest that the reaction product under physiological conditions is a six-coordinate, "base-on" form of the vitamin with a weakly bound alpha -dimethylbenzimidazole base and a bent nitrosyl coordinated to cobalt at the beta -site of the corrin ring. The nitrosyl adduct can formally be described as Cbl(III)-NO-. The kinetics of the binding and dissociation reactions was investigated by laser flash photolysis and stopped-flow techniques, respectively. The activation parameters, DeltaH(double dagger), DeltaS(double dagger), and DeltaV(double dagger), for the forward and reverse reactions were estimated from the effect of temperature and pressure on the kinetics of these reactions. For the "on" reaction of Cbl(II) with NO, the small positive DeltaS(double dagger) and DeltaV(double dagger) values suggest the operation of a dissociative interchange (I-d) substitution mechanism at the Co(II) center. Detailed laser flash photolysis and O-17 NMR studies provide evidence for the formation of water-bound intermediates in the laser flash experiments and strongly support the proposed I-d mechanism. The kinetics of the "off" reaction was studied using an NO-trapping technique. The respective activation parameters are also consistent with a dissociative interchange mechanism.