Membrane-bound DD-carboxypeptidase of the unstable L-form of Proteus mirabilis was solubilized by the non-ionic detergent Genapol X-100 and purified to protein homogeneity by affinity chromatography on ampicillin bound to succinyl-aminododecyl-cellulose. The purified enzyme with a molecular weight of 43000 is inhibited non-competitively by penicillin G and carbenicillin, indicating a function of the penicillins as allosteric inhibitors. Sensitivity of the enzyme to penicillins is only moderate with a Ki of 1 muM for penicillin G. Breakdown of.the enzyme-inhibitor complex EI with different penicillins occurs rapidly with reappearance of active DD-carboxypeptidase. The half-life of EI with penicillin G is 5.5 min at 30 degrees C and 3.5 min at 37 degrees C, 10--1000-fold shorter than EI half-lives of DD-carboxypeptidases in several other bacteria. The low stability of the enzyme-inhibitor complex and the moderate penicillin sensitivity appear to be the basis for the continued activity of DD-carboxypeptidase during growth of the L-form and synthesis of peptidoglycan in the presence of high concentrations of penicillin.