The properties of a rat hepatic microsomal enzyme that hydrolyses O,O-diethyl-p-nitrophenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzyme which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.