A decade ago Jacobsen and co-workers reported the first evidence for the presence of glutathionylcobalamin (GSCbl) in mammalian cells and suggested that it could in fact be a precursor to the formation of the two coenzyme forms of vitamin 1312, adenosylcobalamin and methylcobalamin (Pezacka et al. Biochem. Biophys, Res. Commun. 1990, 169, 443). It has also recently been proposed by McCaddon and co-workers that GSCbl may be useful for the treatment of Alzheimer's disease (McCaddon et al. Neurology 2002, 58, 1395). Aquacobalamin is one of the major forms of vitamin B-12 isolated from mammalian cells, and high concentrations of glutathione (1-10 mM) are also found in cells. We have now determined observed equilibrium constants, K-obs(GSCbl), for the formation of GSCbl from aquacobalamin and glutathione in the pH range 4.50-6.00. K-obs(GSCbl) increases with increasing pH, and this increase is attributed to increasing amounts of the thiolate forms (RS-) of glutathione. An estimate for the equilibrium constant for the formation of GSCbl from aquacobalamin and the thiolate forms of glutathione of similar to5 x 10(9) M-1 is obtained from the data. Hence, under biological conditions the formation of GSCbl from aquacobalamin and glutathione is essentially irreversible. The rate of the reaction between aquacobalamin/hydroxycobalamin and glutathione for 4.50 < pH < 11.0 has also been studied and the observed rate constant for the reaction was found to decrease with increasing pH. The data were fitted to a mechanism in which each of the 3 macroscopic forms of glutathione present in this pH region react with aquacobalamin, giving k(1) = 18.5 M-1 s(-1), k(2) = 28 +/- 10 M-1 s(-1), and k(3) = 163 +/- 8 M-1 s(-1). The temperature dependence of the observed rate constant at pH 7.40 (similar tok(1)) was also studied, and activation parameters were obtained typical of a dissociative process (DeltaH(double dagger) = 81.0 +/- 0.5 kJ mol(-1) and DeltaS(double dagger) = 48 +/- 2 J K-1 mol(-1)). Formation of GSCbl from aquacobalamin is rapid; for example, at similar to5 mM concentrations of glutathione and at 37 degreesC, the half-life for formation of GSCbl from aquacobalamin and glutathione is 2.8 s. On the basis of our equilibrium and rate-constant data we conclude that, upon entering cells, any free (protein-unbound) aquacobalamin could be rapidly and irreversibly converted to GSCbl. GSCbl may indeed play an important role in vitamin B-12-dependent processes.