Penicillins and cephalosporins inhibit the hydrolysis of d-alanyl-β-naphthylamide by aminopeptidase D (EC 3.4.11.—) from Bacillus subtilis. The inhibition is predominantly non-competitive, although the more effective inhibitors, i.e., those with the lower K i values, seem to exhibit a tendency toward competitive kinetics, while penicillin V, the antibiotic with the largest K i , exhibits a strong tendency toward uncompetitive kinetics. The removal of antibiotic from the enzyme by gel filtration on Sephadex G-25 restores 100% activity to the enzyme, and suggests that the inhibition does not derive from a covalent antibiotic-enzyme complex. The antibiotics which have been studied, with their respective K i values (mM) and inhibition type are: methicillin ( 1.01 ± 0.34, mixed non-competitive-competitive); cephaloridine ( 2.43 ± 0.17, non-competitive); cloxacillin ( 3.19 ± 1.29, mixed non-competitive-competitive); oxacillin ( 6.88 ± 0.77, non-competitive); cephalothin ( 8.12 ± 0.99, non-competitive); penicillin G ( 20.0 ± 3.21, non-competitive) and penicillin V ( 32.1 ± 4.90, mixed non-competitive-uncompetitive). An empirical correlation exists between the K i value and the freedom of rotation about the bond between the phenyl ring and the atom alpha to the ring in the variable side chain portion of the penicillin molecule.