Catalytic antibody 43D4-3D12, which was generated against the substituted tertiary amine 1, catalyzes the elimination of HF from beta-fluoroketone 2. We have cloned and produced the antibody as a chimeric Fab and constructed a model of the active site-substrate complex. Mutagenesis studies of the active site indicate that Glu(H)50 acts as the general base and suggest that Tyr96(L) may also play a role in the elimination reaction. Antibody 43D4-3D12 also efficiently catalyzes the elimination of HBr from substrate 4 by an E2 mechanism, again involving selective abstraction of the proton beta-to the nitrophenyl ring by Glu(H)50. The antibody-catalyzed reaction affords predominantly the internal olefins, whereas the major product resulting from the uncatalyzed reaction is the alcohol, which arises from the competing substitution reaction. In addition, antibody 43D4-3D12 catalyzes an acetal hydrolysis reaction in which Glu(H)50 likely acts as a general acid. These studies point to the success of this particular hapten design strategy in generating an active site with a desired catalytic functional group. They also illustrate the utility of using related reactions as mechanistic probes of biological catalysis.