The activity of rat lung epoxide hydrolase (epoxide hydrolase, EC 3.3.2.3) was studied using two lipid epoxides which can be isolated from lung tissue. These epoxides displayed different Km,app and hydration rates. Methyl cis-9,10-epoxystearate was hydrated 20-times more rapidly than cholest-5 alpha,6 alpha-epoxy-3 beta-ol. The Km for the lung microsomal enzyme was variable and dependent on the microsome concentration in the medium. A soluble epoxide hydrolase was also detected in both lung and liver. This enzyme appears similar to the microsomal enzyme in its activity toward methyl epoxystearate. The measured activities for liver microsomal epoxide hydrolase were over 8-times those for lung microsomes; activity against cholesterol epoxide was 40-times greater for liver. In spite of the slow rates measured with cholesterol epoxide in lung preparations, this compound was an effective competitive inhibitor against methyl epoxystearate over a wide concentration range. This suggests that cholesterol epoxide readily binds to epoxide hydrolase and is an effective competitive inhibitor against a much more actively metabolized substrate, methyl epoxystearate. Such circumstances indicate that cholesterol epoxide binds with a high degree of nonproductivity to lung microsomal epoxide hydrolase. This attribute of lung epoxide hydrolase may relate to the relatively high concentrations of cholesterol epoxide found in lung tissue.