Cytidine-3'-phosphate was reacted with p-benzoquinone under neutral aqueous conditions, and the fluorescent product formed was isolated and characterized. The structure of the covalent adduct was identified as (3'-hydroxy)-3,N4-benzetheno-cytidine-3'-phosphate by high-resolution MS and 1H NMR spectroscopy. A similar product was isolated from the reaction of 2'-deoxycytidine-3'-phosphate with a hydroquinone-p-benzoquinone mixture. 32P-Postlabeling of calf thymus DNA reacted with p-benzoquinone detected several adducts, the principal adduct being (3'-hydroxy)-3,N4-benzetheno-2'-deoxycytidine-3'-phosphate. Our studies demonstrate that the reaction of DNA with p-benzoquinone in vitro leads to multiple DNA adducts. 32P-Postlabeling may allow detection of benzene-DNA adducts in vivo.