A modified purification procedure for lysine 2,3-aminomutase from Clostridium subterminale strain SB4 has been developed. The enzyme was shown to be free of α-lysine racemase activity by use of Marfey's reagent. The isotope effect for transfer of the 3-pro-R hydrogen of α-lysine to the 2-pro-R position of β-lysine was determined to be kH/kD= 2.9 ± 0.3 by a competition experiment between L-[3,3-2H2] lysine and L-[2-2H1] lysine. No kinetic isotope effect was detected for α-deuterium substitution of α-lysine. Intermolecular transfer of deuterium was detected in two crossover experiments. Conversion of a mixture of L-[3,3-2H2] lysine and L-[4,4,5,5-2H4] lysine to β-lysine gave a product showing an enhanced M+ 5 peak in the mass spectrum. Similarly a mixture of L-[3,3-2H2] lysine +L-[UL-13C] lysine gave β-lysine having an enhanced M+ 7 peak in the mass spectrum. These results suggest that the hydrogen migration might involve a hydrogen carrier in a manner similar to the mechanisms of adenosylcobalamindependent vicinal interchange reactions. A theory that S-adenosyl-L-methionine might be acting as this hydrogen carrier was tested by co-incubating a mixture of L-[3,3-2H2]lysine and an excess of S-adenosyl-L-methionine. After incubation, the S-adenosyl-L-methionine was degraded to 5′methylthioadenosine, which was shown by 2H n.m.r. to be devoid of deuterium. The results show that S-adenosyl-L-methionine does not serve as hydrogen carrier in the reaction.