Leukotriene A₄ (LTA₄) is a chemically reactive conjugated triene epoxide that is formed by 5-lipoxygenase and is an intermediate in the formation of the biologically active eicosanoids leukotriene B₄ and leukotriene C₄. The present study was undertaken to determine whether or not LTA₄ could serve as an electrophilic species that nucleosides and nucleotides could attack, ultimately resulting in a covalent adduct. Electrospray ionization mass spectrometry and tandem mass spectrometry were used to study the covalent binding of LTA₄ with uridine, cytidine, adenosine, and guanosine. The reaction with guanosine was found to yield five major and at least six minor adduct species. Reversed phase HPLC and mass spectrometric data suggested that the guanosine attacked LTA₄ either at carbon-12 or carbon-6 with opening the epoxide at carbon-5 to yield a series of adducts characterized by the molecular anion [M–H]⁻ at m/z 600.3. Reactions of LTA₄ with mixtures of nucleosides and nucleotides revealed that guanine-containing nucleosides were the most reactive toward LTA₄. The facility of the reaction of guanine with LTA₄ raises the possibility that this intermediate of leukotriene biosynthesis formed on or near the cellular nuclear envelope may react with nucleosides and nucleotides present in RNA or DNA.