The mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by the antibiotic L-cycloserine was investigated. L-Cycloserine is a time-dependent inactivator of GABA aminotransferase; no enzyme activity returns upon gel filtration or dialysis. Treatment of GABA aminotransferase with [C-14]-L-cycloserine, followed by rapid gel filtration, gives enzyme containing 1.1 equiv of radioactivity bound. Dialysis or denaturation by acid base, or urea releases the radioactivity. Inactivation of [H-3]pyridoxal 5＇-phosphate (PLP)-reconstituted GABA aminotransferase with L-cycloserine followed by dialysis or denaturation also leads to the release of radioactivity from the enzyme. Both the released [C-14]- and [H-3]-labeled adducts comigrate by HPLC, suggesting that the inactivation adduct is a condensation product of L-cycloserine with the PLP coenzyme. By HPLC comparison, it was shown that the radiolabeled adduct is not PLP, PMP, PLP oxime, or 4-[3-hydroxy-2-methyl-5-(phosphooxymethyl)-4-pyridinyl]-2-oxo-3-butenoic acid (20), the expected product of an enamine-type inactivation mechanism. On the basis of the stability of the released adduct to acid and base and its UV-visible spectrum, which has the appearance of a PMP analogue, a simple Schiff base between PLP and cycloserine also was excluded. HPLC of the cycloserine-coenzyme adduct had a retention time very similar to that of the gabaculine-coenzyme adduct. Electrospray ionization tandem mass spectrometry of the isolated cycloserine-coenzyme adduct is consistent with a structure that is one of the tautomeric forms of the Schiff base between PMP and oxidized cycloserine (21).