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Pseudomonas aeruginosa is an opportunistic bacterium which causes serious infections in immunocompromised and cystic fibrosis patients (10). As with many gram-negative bacteria, P. aeruginosa produces acyl-homoserine lactone (AHL) quorum-sensing (QS) signaling molecules termed autoinducers which allow the single-celled organisms to coordinate their actions (36). N-(3-Oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a key autoinducer synthesized by P. aeruginosa which regulates the expression of extracellular virulence factors and biofilm formation (5, 36). Rats and mice experimentally infected with P. aeruginosa mutants deficient in the ability to produce or respond to 3OC12-HSL exhibited significantly diminished lung pathology, bacterial dissemination, and morbidity and accelerated bacterial clearance compared to animals infected with wild-type bacteria, demonstrating the importance of 3OC12-HSL for P. aeruginosa pathogenicity (14, 21, 27, 31, 40). 3OC12-HSL also has an array of immunomodulatory effects on eukaryotic cells, including the induction of apoptosis, inhibition of leukocyte proliferation, activation of neutrophils and macrophages, and induction of proinflammatory mediators (7, 15, 34, 37, 39, 43). Recently, it was shown that a number of mammalian cell lines were able to inactive 3OC12-HSL (5), providing a possible mechanism for reduction of bacterial virulence. Mammalian paraoxonases (PONs) are a unique, highly conserved family of calcium-dependent esterases consisting of PON1, PON2, and PON3 (8). Human PON1 and PON3 are synthesized predominantly in the liver from where PON1 and some PON3 are secreted into the blood and associated with high-density lipoproteins (HDL) (29). PON2 is not in serum but is expressed in many tissues and cell types (8, 25). PONs exhibit antioxidative properties and afford protection from atherosclerosis in mouse models; however, the mechanisms by which they mediate these properties are not yet established (1, 16, 24, 32, 33). PONs hydrolyze a broad range of esters, including phosphotriesters, arylesters, and lactones, and have overlapping, but also distinct, substrate specificities (9). Although the physiological function(s) and natural substrates for the PONs are uncertain, accumulating evidence indicates that the lactonase activity of the PONs may be its natural function (9, 17). Serum PON1 hydrolyzes the lactone ring of 3OC12-HSL (26) and the lactonase activity of the PONs extends over a number of AHL QS compounds with various acyl chain lengths (9, 38). Sera from PON1 knockout (PON1−/−) mice are deficient in 3OC12-HSL hydrolytic activity, but surprisingly, PON1−/− mice had a higher rate of survival than wild-type mice after intraperitoneal injection of the bacterium P. aeruginosa (26). PON2 and PON3 were shown to be up-regulated in the PON1−/− mouse airway epithelium, and it was suggested that this up-regulation may lead to increased inactivation of 3OC12-HSL. However, the possible up-regulation and contribution of other enzymes toward 3OC12-HSL inactivation in this model were not investigated. For murine tracheal epithelial cells, PON2 was shown to be important for the inactivation of 3OC12-HSL by demonstrating that the cell lysates from PON2-deficient mice had an impaired ability to hydrolyze 3OC12-HSL and that P. aeruginosa QS was enhanced in these epithelial cell cultures (38). Interestingly, in intact epithelial cells there was no difference in the rates of 3OC12-HSL inactivation between the wild-type and PON2-deficient cells. Thus, the relative importance of the role that PON2 plays in the intact cells is not clear, and other inactivation pathways, PON or non-PON, may be important for 3OC12-HSL inactivation in these cells. Mammals express a broad range of enzymes, such as carboxylesterases, amidases, acylases, proteases, oxidases, and reductases, which could potentially inactive 3OC12-HSL, as well as other AHLs, and the importance of PON-mediated inactivation of 3OC12-HSL relative to other enzymatic pathways is not known. Therefore, the aim of this study was to determine if there are other mammalian enzymes which may inactivate 3OC12-HSL and to evaluate the contribution of the PONs to 3OC12-HSL metabolism. We found that the human PONs, particularly PON2, could efficiently hydrolyze 3OC12-HSL. PONs were the major enzymes inactivating this lactone in human and mouse serum, mouse lung and liver homogenates, and cultured human cell lysates. Thus, our study suggests that the PONs, especially PON2, may represent a key defense mechanism against the P. aeruginosa QS autoinducer 3OC12-HSL.