L-Leucinal, prepared by enzymatic oxidation of L-leucinol with alcohol dehydrogenase, is found to be a very strong competitive inhibitor of porcine kidney aminopeptidases. For the enzyme from kidney microsomes acting on L-leucine p-nitroanilide (Km = 5.2 x 10(-4) M), for Ki for L-leucinal was 7.6 x 10(-7) M at pH 7.2 and 25 degrees C. For the enzyme from kidney cytosol acting on L-leucine p-nitroanilide (Km = 7.7 x 10(-4) M), Ki for L-leucinal was 6 x 10(-8) M; Ki for glycinal (analogous to glycine derivatives that are poor substrates) was 6.8 x 10(-4) M. In dilute aqueous solution, leucinal exists in unfavorable equilibrium with its covalent hydrate, whose concentration exceeds that of the free aldehyde by a factor of 40. The affinity of the enzyme for the free aldehyde is correspondingly greater than its Ki values would suggest, exceeding the apparent affinity of the substrate by a factor of about 10(6). A comparison of binding affinities suggests that L-leucinal forms an inhibitory complex analogous in structure to unstable intermediates and substrate transformation by leucine aminopeptidase, and strengthens the likelihood that this enzyme may act by a double-displacement mechanism.