The enzyme 2-keto-3-deoxygluconate-6-P aldolase of Pseudomonas putida is inactivated by one of the chiral forms of 2-keto-(3RS)-3-bromobutyric acid (bromoketobutyrate). The inactivation shows saturation kinetics and competition with pyruvate. The minimal inactivation half-time is 4 min and that concentration of bromoketobutyrate half-saturating the enzyme is 2 mM. (3RS)-[3-3H]bromoketobutyrate is catalytically detritiated during enzyme inactivation. A kinetic analysis of rates gave data consistent with both catalysis and inactivation occurring at a single protein site, the catalytic site. The enzyme only detritiates one of the two optical isomers of bromoketobutyrate, and that form which is detritiated also alkylates the catalytic site. The inactive isomer of reagent degrades, with inversion, to L-lactate so that the chiral form specific for the enzyme is 2-keto-(3S)-3-bromobutyrate. Thus, as is the case with bromopyruvate, the enzyme catalyzes protonation of the re face at C-3 of the enzyme-reagent eneamine. As a result, bromoketobutyrate could serve as a chiral probe for stereochemical constraints of selected pyruvate-specific lyase active sites.