Abstract Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3′ to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1. The residual modified dinucleoside monophosphates obtained from a nuclease P1 (plus acid phosphatase) digest of DNA can be 32P-postlabeled using T4 polynucleotide kinase. Using this assay the formamido single lesion and the formamido–8-oxoguanine double lesion were detected in calf thymus DNA after X-irradiation in oxygenated aqueous solution. The lesions were measured in the forms d(PFpG) and d(PFpGH), where PF stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and GH stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/μg DNA, respectively.