A mutant of Escherichia coli that is deficient in D-alanine carboxypeptidase IA has been isolated. The enzyme is membrane bound and moderately sensitive to penicillin. It catalyzes in vitro both D-alanine carboxypeptidase and transpeptidase reactions. Being able to synthesize crosslinked peptidoglycan both in vivo and in vitro despite the absence of enzyme activity, the newly isolated mutant grew normally under a wide range of growth conditions. Therefore, this enzyme, like D-alanine carboxypeptidase IB, is not required for normal peptidoglycan synthesis in E. coli. The defect in the activity of D-alanine carboxypeptidase IA in the mutant however was not associated with disappearance of penicillin-binding proteins 5 and 6 (which have been shown to be D-alanine carboxypeptidase IA) or any of the other protein bands that bind [14C]penicillin G. Genetic mapping studies showed that the mutation (dacA) is located close to leuS(13.7 min) on the E. coli chromosome map. Double mutants (dacA dacB) that are deficient in both D-alanine carboxypeptidases IA and IB were obtained. These double mutants also were found to grow normally and to catalyze normal formation of crosslinked peptidoglycan.