The D-alanine carboxypeptidase of Bacillus subtilis is a particulate enzyme that is irreversibly inactivated by penicillins and cephalosporins. However, the lethal concentrations of these antibiotics are not the same as those that inhibit enzymatic activity in vitro. 6-Aminopenicillanic acid inactivates at least 95% of the enzyme at nonlethal concentrations. Conversely, cephalothin is lethal at concentrations that do not inactivate the enzyme. Experiments with intact, growing cells confirm the results obtained in vitro. Therefore, a killing site distinct from the carboxypeptidase must be postulated.