Leukotriene E4 was metabolized to two polar products by rat liver microsomes. These products were characterized by physico-chemical and chemical techniques. The chemical structures, (5S, 6R)-5,20-dihydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraenoic acid (omega-hydroxy-leukotriene E4) and (5S, 6R)-5-hydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraen-1,20-d ioic acid (omega-carboxy-leukotriene E4) suggested that leukotriene E4 was transformed by an omega-hydroxylase and omega-hydroxyleukotriene E dehydrogenase in sequence. N-Acetyl-leukotriene E4 was also transformed by these enzymes, but at a rate six times lower than leukotriene E4. The products formed from N-acetylleukotriene E4 were characterized as being N-acetyl-omega-hydroxy-leukotriene E4 and N-acetyl-omega-carboxy-leukotriene E4. Other substrates were 11-trans-leukotriene E4 and N-acetyl-11-trans-leukotriene E4. In contrast, leukotrienes C4 and D4 were not converted into omega-oxidized metabolites. The leukotriene E omega-hydroxylase reaction required NADPH and molecular oxygen as cofactors, and was most rapidly catalyzed by liver microsomes. Liver cytosol, fortified with NAD+, converted omega-hydroxyleukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4 into omega-carboxy metabolites. Microsomes contained at least 18 times less omega-hydroxy-leukotriene E dehydrogenase activity than did cytosol. Liver microsomes supplemented with acetyl-coenzyme A converted omega-hydroxy and omega-carboxy-leukotriene E4 into the corresponding N-acetyl derivatives. The novel enzyme, leukotriene E omega-hydroxylase, which is described here is distinct from a previously described leukotriene B omega-hydroxylase based on substrate competition and kinetic data.