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The d-glutamate-adding and d-alanyl-d-alanine-adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54000 ± 2000 and 51000 ± 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the d-Glu-adding enzyme, DDP-MurNAc-l-Ala (DDP = dihydrouridine 5′-diphosphate) and P1-MurNAc-l-Ala were substrates of the reaction. In the case of the d-Ala-d-Ala-adding enzyme, only DDP-MurNAc-l-Ala-d-Glu(-meso-A2pm) was a subsrate; P1-MurNAc-l-Ala-d-Glu(-meso-A2pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the d-Glu-adding enzyme, even closely related analogues of d-glutamate hardly inhibited the reaction.