The substrate specificity of NAD-dependent formate dehydrogenase from the methylotrophic bacterium Achromobacter parvulus T1 was studied. The kinetic mechanism of S-formyl glutathione oxidation was determined. The initial velocity studies and inhibition analysis were carried out. It was shown that the kinetic mechanism for the enzyme with S-formyl glutathione as a substrate is similar to that with formate and is rapid-equilibrium random. Using independent methods, it was found that formate dehydrogenase forms a binary complex with S-formyl glutathione (Kd = 2.5 mM).