Rat liver homogenates catalyzed the elimination of fluoride from (R,S)-alpha-fluoro-beta-alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial L-alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with L-alanine and glyoxylate as substrates, copurified with the alpha-fluoro-beta-alanine-defluorinating enzyme. The NH2-terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of L-alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of kcat = 17 sec-1, KL-Ala = 3.2 mM, and Kglyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of (R)-alpha-fluoro-beta-alanine and (S)-alpha-fluoro-beta-alanine were kcat = 6.2 and 2.6 min-1, respectively, and Km = 2.7 and 0.88 mM, respectively. L-Alanine potently inhibited the defluorination reaction with an apparent Ki of 0.024 mM. (R,S)-alpha-Fluoro-beta-alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridoxamino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with (R,S)-alpha-fluoro-beta-alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of (R,S)-alpha-fluoro-beta-alanine but was inactivated completely during dehalogenation of beta-chloro-L-alanine.