An antibiotic,d-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) andd-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designatedalrSandddlS, respectively, from DCS-producingStreptomyces lavendulaeATCC25233. Each gene product was purified to homogeneity and characterized.Escherichia coli, transformed with a pET vector carryingalrSorddlS, displays higher resistance to DCS than the same host carrying theE. coliALR- or DDL-encoded gene inserted into the pET vector. Although theS. lavendulaeDDL was competitively inhibited by DCS, theKivalue (920 μm) was obviously higher (40∼100-fold) than those forE. coliDdlA (9 μm) or DdlB (27 μm). The highKivalue of theS. lavendulaeDDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for theS. lavendulaeALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of theE. coliALR. We conclude that ALR from DCS-producingS. lavendulaeis also one of the self-resistance determinants.