The stereochemical course of dehydrogenation catalyzed by acyl-CoA oxidase was investigated using the enzymes from rat liver peroxisomes and Candida lipolytica. Stearoyl-CoA and nonanoyl-CoA, stereospecifically labeled with deuterium at either C-2 or C-3, were incubated with the enzyme, the products were converted to methyl esters and their deuterium contents were measured by gas chromatography-mass spectrometry. The results suggested that acyl-CoA oxidase-catalyzed dehydrogenation occurred by anti-elimination of the pro 2R and pro-3R hydrogens of acyl-CoA.