Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids, the first step in the biosynthesis of a large group of biologically active fatty acid metabolites collectively named oxylipins. In the present study we report the characterization of the enzymatic activity of the six lipoxygenases found in the genome of the model plant Arabidopsis thaliana. Recombinant expressed AtLOX-1 and AtLOX-5 had comparable oxygenase activity with either linoleic acid or linolenic acid. AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 displayed a selective oxygenation of linolenic acid. Analyses by high-performance liquid chromatography and gas chromatography-mass spectrometry demonstrated that AtLOX-1 and AtLOX-5 are 9S-lipoxygenases, and AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 are 13S-lipoxygenases. None of the enzymes had dual positional specificity. The determined activities correlated with that predicted by their phylogenetic relationship to other biochemically-characterized plant lipoxygenases.