The present study was aimed at the characterization of the major adducts formed by reaction of the metabolites of [14C]benzene with rat hemoglobin in vivo. Groups of 12-week-old male Fisher rats received i.p. injections of a single dose of 10 mmol/kg body weight or three equal daily subdoses of 3.3 mmol/kg body weight of [14C]benzene. High-performance liquid chromatographic analysis of strong acid hydrolysates of the [14C]benzene-modified globin indicated that the two major adducts in rats cochromatographed with synthetic S-(2,5-dihydroxyphenyl)cysteine and S-phenylcysteine. These adducts were converted to O,O'-S-tris-acetyl-3-thiol-hydroquinone and S-phenylthioacetate, which were then characterized by gas chromatography/mass spectrometry. The major radioactive adduct peaks accounted for 60-75% of the total radioactivity associated with rat globin. Characterization of the S-(2,5-dihydroxyphenyl)cysteine adduct provides evidence that p-benzoquinone is formed as a reactive metabolite of benzene. Formation of the S-phenylcysteine adduct indicates that benzene oxide and/or a hydroxycyclohexadienyl free radical is formed as an active intermediate upon i.p. injection of benzene into rats.