Four facultative anaerobes were isolated from soil as producers of 3-methylaspartate ammonia-lyase (MAL). They were identified as Citrobacter sp. No. 0504, Proteus sp. No. 0601, Escherichia coli No. 1002, and Enterobacter sp. No. 1202. The enzyme activities were induced when the strains were cultivated anaerobically or statically in the presence of (S)-glutamic acid or (2S, 3S)-3-methylaspartic acid. Using cell-free extracts of the isolates, several 3-substituted (S)-aspartic acid derivatives such as (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and (2R,3S)-3-chloroaspartic acid were synthesized in optically pure form from mesaconic acid, ethylfumaric acid, and chlorofumaric acid, respectively.