The existence of two or more enzyme forms with paraoxonase activity has been reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxonase activity (M1 and M2) after non-specific affinity chromatography of rat liver microsomes on Cibacron Blue 3GA. The first peak (M1) was obtained during the washing of the column and coeluted with albumin. The second active peak (M2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparison of both active fractions on the basis of kinetic parameters, heat inactivation and pH stability, calcium requirement and inhibition by EDTA and several metals was performed. Our results support the fact that two proteins capable of hydrolyzing paraoxon are present in rat liver microsomes. Furthermore, during the purification to homogeneity of rat liver paraoxonase we have performed a study of its hydrolytic ability against three different substrates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profile in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation and effect of inhibitors were used as identity criteria for the three hydrolytic activities. Our results show evidence for the hydrolysis of paraoxon and phenylacetate by the same protein from rat liver (paraoxonase).