Our knowledge about the metabolism of alkylcyclobutanones (2-ACBs) is limited, and the lack of literature on the metabolism of 2-ACBs causes consumers to doubt the safety of irradiated foods. The objectives of this study were to evaluate the metabolism of 2-dodecylcyclobutanone (2-DCB) and identify any possible metabolite. The 2-DCB was mixed with rat S9 (postmitochondrial supernatant fraction) and beta-nicotinamide adenine dinucleotide phosphate (NADPH) in phosphate buffer (pH 7.4) and incubated for 2 h at 37 degrees C. Then, the incubation mixture was mixed with sodium sulfate and extracted with n-hexane by using a Soxhlet apparatus. The hexane extract was concentrated under nitrogen and injected into the gas chromatography-mass spectrometry (GC-MS) machine running in selective ion monitoring mode (SIM) to measure 2-DCB concentration. The hexane extract from the in vitro and in vivo studies was also derivatized with a silylation reagent and injected into a GC-MS running in full scan mode. The average percentage of 2-DCB recovered from the test incubations was 23%, compared with 50% from the controls. The GC-MS chromatograms of the derivatized samples showed a unique peak in the in vitro test incubations and in the hexane extract of the rat feces that were given 2-DCB. This peak was later identified as 2-doecylcyclobutanol.