Papain is strongly inhibited by aldehydes resembling carboxylic acids, released by hydrolysis of specific substrates (Westerik, J. O'C., and Wolfenden, R. (1972), J. Biol. Chem. 247, 8195-8197). Inhibitory complexes might involve binding of the aldehyde intact or as a covalent hydrate, or the aldehyde might undergo covalent addition of an active site sulfhydryl group to form a thiohemiacetal derivative. In an attempt to distinguish between these possibilities, benzamidoacetaldehyde-1-d has been synthesized, and its properties compared with those of the undeuterated inhibitor. After correction for differences in hydration, the observed effect on inhibition is found to be compatible with formation of a thiohemiacetal. In keeping with this conclusion, benzamidoethanol (a partial analogue of the covalent hydrate) and benzamide, N-methylbenzamide and N-ethylbenzamide (somewhat similar to the free aldehyde in size and hydrophobic character) are found to exhibit negligible affinity for the active site.