The paraoxonase (PON1) enzyme exhibits several enzymatic activities, including an arylesterase activity, and it is thought to inhibit lipoprotein peroxidation and facilitate reverse cholesterol transport. Reduced arylesterase activity has been associated with several chronic diseases including hypercholesterolemia. An accurate and reproducible determination of arylesterase activity may be useful for predicting the risk of cardiovascular disease. We present a new method to measure serum arylesterase activities in rat and mouse serum activity using a simulated body fluid (SBF). To validate the SBF method in rodents, arylesterase activity was measured in 20 mice (age 6-18 months) and 20 rats, 10 of which were hypercholesterolemic. When compared with the reference Tris/HCl method, the linearity range of arylesterase activity was higher using SBF indicating its potential use in a larger concentration interval. Furthermore, the measurements differed markedly in samples with very low arylesterase activity due to the tendency of the Tris/HCl method to overestimate arylesterase activity in the low range. The ionic environment in SBF, which is physiologically related to serum, is likely to contribute to the improved measurement of arylesterase activity. (C) 2008 Elsevier Inc. All rights reserved.