This article reviews the scientific literature on the application of alpha-ketoheterocycles to the discovery of potent enzyme inhibitors. The alpha-ketoheterocycle functionality provides a moderately electrophilic ketone carbonyl with 'tunable' reactivity, as well as a structural template for introducing new interactions in the enzyme active-site cleft. This type of moiety has served an important role in the design of active-site-directed inhibitors of diverse serine and cysteine proteases, and of fatty acid amide hydrolase (FAAH). Potent inhibitors have been identified for, inter alia, elastase, thrombin, factor Xa, tryptase, chymase, cathepsin K, cathepsin S, and FAAH. For example, 6e is an orally active inhibitor of human neutrophil elastase that entered human clinical studies, 52h is an orally bioavailable inhibitor of human chymase, and 82m is a FAAH inhibitor with in vivo endocannabinoid-enhancing activity.