Oxidative stress is a cardinal manifestation of various intestinal disorders. However, very little knowledge is available on the intestine's inherent defense mechanisms against free radicals. This study was designed to determine the protein expression, subcellular localization and oxidative stress response of paraoxonase2 (PON2), a member of a powerful antioxidant family in human and rat intestine. Biochemical and ultrastructural experiments all showed a substantial expression of PON2 in human and rat intestine. Western blot analysis disclosed higher levels of PON2 in the jejunum than in the duodenum, ileum and colon. Cell fractionation revealed a predominant PON2 association with microsomes and lysosomes in the human jejunum, which differed from that in rats. PON2 was detected in the intestine as early as week 15 of gestation and was significantly increased by week 20. Iron/ascorbate-mediated lipid peroxidation induced a marked decrease in PON2 expression in intestinal specimens coincidental to an abundant rise in malondialdehyde (MDA). On the other hand, pre-incubation with potent antioxidants, such as butylated hydroxytoluene, trolox and N-acetylcysteine, prevented iron/ascorbate-generating PON2 reduction in parallel with MDA suppression. Finally, the pre-incubation of permeabilized Caco-2 cells with purified PON2 led to a protection against iron/ascorbate-induced lipid peroxidation. These observations demonstrate that the human intestine is preferentially endowed with a marked PON2 expression compared with the rat intestine and this expression shows a developmental and intracellular pattern of distribution. Furthermore, our observations suggest PON2 protective effects against pro-oxidant stimuli in the small intestine.