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D-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-alanine racemase (Alr) and Dalanine: D-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Alr in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Alr and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Alr by DCS decreases the intracellular pool of D-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts of L- and D-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess of L- over D-alanine. In all strains, DCS treatment led to a significant accumulation of L-alanine and a concomitant decease of D-alanine, with approximately a 20-fold excess of L-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Alr. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-alanine branch of mycobacterial peptidoglycan biosynthesis.