The specificity of L-methionine-gamma-lyase with respect to the stereochemical structure of the thiol substrate in the gamma-substitution reaction has been demonstrated. Cells of Citrobacter intermedius containing L-methionine-gamma-lyase catalyze the exchange reactions between L-methionine and 2-propylthiol or 2-butylthiol which leads to the formation of s-2-propylhomocysteine and s-2-butylhomocysteine, respectively. The yields of these products are comparable to the yield of s-butylhomocysteine in the reaction of normal butylthiol with L-methionine, thus 2-propylthiol and 2-butylthiol are effective substrates of L-methionine-gamma-lyase. On the other hand in the reaction of 3-pentylthiol, only traces of the expected product, s-3-pentylhomocysteine, were formed and in the case of 2-methyl-2-butylthiol, the expected product of gamma-substitution, s-2-methyl-2-butylhomocysteine, was not formed at all. In the reaction with racemic 2-butylthiol, only one diastereomer of s-2-butylhomocysteine was obtained. The unreacted 2-butylthiol isolated after the reaction catalyzed by partially purified preparation of L-methionine-gamma-lyase was enriched with (R)-enantiomer which indicated the preferential reaction of the (S)-enantiomer.