A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.