The penicillin-sensitive D-alanine carboxypeptidases of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus catalyzed the hydrolysis of the D-lactic acid residue from the depsipeptide diacetyl-L-lysyl-D-alanyl-D-lactic acid. The ester substrate was hydrolyzed faster than the peptide analogue, diacetyl-L-lysyl-D-alanyl-D-alanine, by the B. subtilis (15-fold) and E. coli (4-fold) carboxypeptidases, presumably because acylation (k(2)), which is the rate-limiting step of the peptidase reaction, occurred more rapidly during cleavage of the ester bond than during cleavage of the amide bond. No rate acceleration was observed with the S. aureus carboxypeptidase for which deacylation (k(3)) is already the rate-determining step with the peptide substrate. The efficiency of utilization of the depsipeptide (V(max)/K(m)) was greatly enhanced (19- to 147-fold) for all three enzymes. After incubation of the B. subtilis carboxypeptidase and [(14)C]diacetyl-L-lysyl-D-alanyl-D-lactic acid at pH 5.0 and lowering of the pH to 3.0, a radioactive acyl-enzyme intermediate containing 0.43 mol of substrate per mol of enzyme was isolated by Sephadex G-50 chromatography. After acetone precipitation, the acyl group of the denatured acyl-enzyme complex appeared to be bound to the protein by an ester bond. Acyl enzymes were also detected for the S. aureus and E. coli carboxypeptidases after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography of enzyme incubated with [(14)C]depsipeptide and precipitated with acetone.