As a class of linoleic acid oxidation products, epoxyketooctadecenoic acids (EKODEs), are formed in vivo and in vitro by a free radical mechanism initiated by either enzymatic or nonenzymatic pathways. They have so far been made available in small-scale quantities, often as isomeric mixtures, from reductive decomposition of linoleic acid-derived hydroperoxides. There is major interest in these compounds owing to their highly potent biological activities and their ability to covalently modify proteins. The synthesis of six EKODE regio- and stereoisomers, two trans alpha',beta'-epoxy-alpha,beta-enones, and two trans and the two cis gamma,delta,-epoxy-alpha,beta-enones was accomplished, with the key steps being Wittig-type reactions and aldol condensations. All six EKODE isomers were confirmed by HPLC to be generated in the autoxidation of linoleic acid promoted by Fe(II)/ascorbic acid through spiking in of authentic samples. On the basis of evidence for EKODE modification of protein His residues, the reactions of Nalpha-benzoyl-L-histidine with autoxidizing linoleic acid and with the individual EKODE isomers were compared, as were the kinetics of the various EKODE reactions with imidazole nucleophiles. The structures of His-EKODE-(E)-I adducts were confirmed to reflect conjugate addition (epoxide ring remains intact) through an NMR study of the reaction of imidazole with a generic EKODE-(E)-I analog. The synthesis of the EKODE isomers makes these important molecules available for further chemical and biological evaluation.