Serum paraoxonase (PON1) is a lipolactonase that associates with HDL-apolipoprotein A-I (HDL-apoA-I) and thereby plays a role in the prevention of atherosclerosis. Current sera tests make use of promiscuous substrates and provide no indications regarding HDL-PON1 complex formation. We developed new enzymatic tests that detect total PON1 levels, irrespective of HDL status and R/Q polymorphism, as well as the degree of catalytic stimulation and increased stability that follow PON1's tight binding to HDLapoA-I. The tests are based on measuring total PON1 levels with a fluorogenic phosphotriester, measuring the lipolactonase activity with a chromogenic lactone, and assaying the enzyme's chelator-mediated inactivation rate. The latter two are affected by tight HDL binding and thereby derive the levels of the serum PON1-HDL complex. We demonstrate these new tests with a group of healthy individuals (n 5 54) and show that the levels of PON1-HDL vary by a factor of 12. Whereas the traditionally applied paraoxonase and arylesterase tests weakly reflect PON1-HDL levels (R 5 0.64), the lipolactonase test provides better correlation (R 5 0.80). These new tests indicate the levels and activity of PON1 in a physiologically relevant context as well as the levels and quality of the HDL particles with which the enzyme is associated. Serum paraoxonase (PON1) is a HDL-associated enzyme playing an important role in organophosphate detoxification and the prevention of atherosclerosis (1, 2). HDLbound PON1 inhibits LDL oxidation (3-5) and stimulates cholesterol efflux from macrophages (6, 7). PON1 knockout mice are highly susceptible to atherosclerosis (8, 9). Accordingly, serum PON1 levels seem to be inversely related to the level of cardiovascular disease, although this correlation is weak (10-12). PON1 hydrolyzes a broad range of substrates and has been traditionally described as paraoxonase/arylesterase (2). However, it recently became apparent that PON1 is in fact a lactonase with lipophilic lactones constituting its primary substrates (13,14). Impairing the lactonase activity of PON1, through mutations of its catalytic dyad (15), diminishes PON1's ability to prevent LDL oxidation and stimulate macrophage cholesterol efflux, indicating that the antiatherogenic functions of PON1 are likely to be mediated by its lipolactonase activity (16).PON1 is synthesized in the liver and secreted into the blood, where it associates with HDL complexes carrying apolipoprotein A-I (apoA-I) (17). The structural model of PON1 indicated that the HDL surface lies in close proximity to PON1's active site, thus providing an optimal environment for the enzyme's interaction with its lipophilic substrates (18). Indeed, it has been shown that PON1 binds HDL-apoA-I particles with nanomolar affinity (19). HDLapoA-I binding stabilizes the enzyme and selectively stimulates its lipolactonase activity (19).The impact of PON1 on atherosclerotic disease and resistance to organophosphate toxicity led to intensive investigations of its natural polymorphism...