Abstract It has been proposed that 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to ethylene via N-hydroxyl-ACC as an intermediate. However, due to its chemical instability the putative intermediate has never been isolated. Here, we have shown that a purified recombinant ACC oxidase can utilize α-aminoisobutyric acid (AIB), an analog of ACC, as an alternative substrate, converting AIB into CO2, acetone, and ammonia. We chemically synthesized the putative intermediate compound, N-hydroxyl-AIB (HAIB), and tested whether it serves as an intermediate in the oxidation of AIB. When [1-14C]AIB was incubated with ACC oxidase in the presence of excess unlabeled HAIB as a trap, no labeled HAIB was detected. By comparing the acetone production rates employing HAIB and AIB as substrates, the conversion of HAIB to acetone was found to be much slower than that of using AIB as substrate. Based on these observations, we conclude that ACC oxidase does not catalyze via the N-hydroxylation of its amino acid substrate. ACC oxidase also catalyzes the oxidation of other amino acids, with preference for the d-enantiomers, indicating a stereoselectivity of the enzyme.