t-4-Hydroxy-2-nonenal (HNE) is a free radical-mediated oxidation product of polyunsaturated fatty acids. As an electrophile, HNE readily binds to proteins and yields diastereomeric cyclic 1,N²-propano adducts with deoxyguanosine (dG). Here, we report the detection and identification of the HNE-derived cyclic 1,N²-propano-dG adducts as endogenous DNA lesions in tissues of untreated rats and humans using a highly sensitive ³²P-postlabeling method in conjunction with high-performance liquid chromatography. These adducts were first verified by their comigration with the synthetic UV standards of HNE-dG adducts. Subsequently, their identities were unequivocally established by two independent reactions. An ∼37-fold increase in the levels of HNE-dG adducts was observed in the liver DNA of F344 rats after treatment with CCl₄, suggesting that tissue lipid peroxidation is a likely source of their formation. Our studies in vitro further indicate that ω-6 polyunsaturated fatty acids are likely a unique class of fatty acids involved in HNE-dG adduct formation.