The adenosylcobalamin-dependent enzyme glutamate mutase from Clostridium tetanomorphum catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3- methylaspartate . In this conversion 6 carbon centers are involved. The stereochemistry of 4 has been elucidated whereas the formation of the methyl group from the methylene group remains to be established. To solve this problem, (2S,3R)- and (2S,3S)-[3,3-2H1,3H]glutamates were prepared via the 2-oxo[3,3-2H2 or 3H] glutarates by incubation with isocitrate dehydrogenase in deuterium oxide or tritiated water. The labelled glutamates were fermented with growing cells of C. tetanomorphum to butyrate and acetate. Butyrate was further degraded to acetate in which methyl group over 90% of the tritium of the starting glutamate was retained. The chirality of the acetates was determined with malate synthase and fumarase. In both samples complete racemisation was found. This result confirms the rule that racemisation occurs in all adenosylcobalamin-dependent rearrangements in which methyl groups are formed. A methylene radical as intermediate could explain these observations. In a control experiment inversion of configuration in the formation of the methine group of (2S,3S)-3-methylaspartate from the methylene group of (2S)-glutamate was confirmed. Glutamates stereospecifically labelled at C-4 were synthesized from chiral acetates via citrate.