Glutathione induces the rapid isomerization of (Z,Z)-muconaldehyde to (E,E)-muconaldehyde via (E,Z)-muconaldehyde, probably via reversible Michael addition of the thiol to one of the enal moieties of the muconaldehyde. Reactions of (E,E)-muconaldehyde with glutathione (in the presence and absence of equine glutathione S-transferase), phenylmethanethiol, N-acetyl-l-cysteine, and N-acetyl-l-cysteine methyl ester were investigated using mass spectrometric techniques. In each case, evidence was obtained for the formation of Michael adducts, e.g., reaction between (E,E)-muconaldehyde and glutathione gave 4-glutathionyl-hex-2-enedial and 3,4-bis-glutathionyl-hexanedial. These experiments suggest that (Z,Z)-muconaldehyde, a putative metabolite of benzene, could lead to the long established urinary metabolite of benzene, (E,E)-muconic acid, via glutathione-mediated isomerization to (E,E)-muconaldehyde.