Leukotriene A4 hydrolase from the human lung was purified to apparent homogeneity. The molecular weight (68,000-71,000), the amino acid composition, and the N-terminal amino acid sequence were similar to those of the human neutrophil enzyme but different from those of human erythrocyte enzyme. The lung enzyme was inactivated by its substrate, leukotriene A4. To elucidate the substrate and the inactivator specificity of this enzyme, we synthesized various geometric and positional isomers of leukotriene A4. 14,15-Leukotriene A4, leukotriene A4 methyl ester, and geometric isomers of leukotriene A4 could not serve as substrates, but they inactivated the enzyme. On the other hand, styrene oxide and (5S)-trans-5,6-oxide-8,10,14-cis-12-trans-eicosatetraenoic acid neither served as substrates nor inactivated the enzyme. These results indicate that whereas allylic epoxide structures of arachidonic acids are responsible for inactivation of the enzyme, the free carboxylic acid, 5,6-oxide, and the tetraene structure with the 7,9-trans-11,14-cis configuration are required as a substrate for leukotriene A4 hydrolase.