Publisher Summary The chapter presents a study on purification and characterization of human lung leukotriene (LT) A4 hydrolase. LT A4 hydrolase (EC 18.104.22.168) catalyzes enzymatic hydration of LTA4 [5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid] to LTB4 [5(S), 12 (R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], a potent chemotactic substance. This reaction on the epoxide differs from that of epoxide hydrolases (EC 22.214.171.124). The latter results in the synthesis of vicinal alcohols (glycol), while the former synthesizes a compound that possesses two hydroxy groups at C-5 and C-12 with a conjugated triene in between. LTA4 hydrolase activity was reported mainly in blood cells, especially polymorphonuclear leukocytes and erythrocytes, and the enzymes from these sources were purified. The enzyme activity is, however, ubiquitously distributed in the cytosolic fraction of various organs of the guinea pig, and higher activities are observed in small intestine, lung, and aorta. To compare the lung enzyme with those found in blood cells, and for further characterization, the enzyme was purified from the human lung. Recently, this enzyme has also been purified from the guinea pig liver and lung. The chapter discusses the preparation and purification of LTA4 methyl ester, saponification of LTA4 methyl ester, assay of LTA4 hydrolase, and several related concepts.