Acetaldehyde is a mutagen and carcinogen which occurs widely in the human environment, sometimes in considerable amounts, but little is known about its reactions with DNA. In this study, we identified three new types of stable acetaldehyde DNA adducts, including an interstrand cross-link. These were formed in addition to the previously characterized N2-ethylidenedeoxyguanosine. Acetaldehyde was allowed to react with calf thymus DNA or deoxyguanosine. The DNA was isolated and hydrolyzed enzymatically; in some cases, the DNA was first treated with NaBH3CN. Reaction mixtures were analyzed by HPLC, and adducts were isolated and characterized by UV, 1H NMR, and MS. The major adduct was N2-ethylidenedeoxyguanosine (1), which was identified as N2-ethyldeoxyguanosine (7) after treatment of the DNA with NaBH3CN. The new acetaldehyde adducts were 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-α]pu rine-10(3H)one (9), 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)- 6-methylpyrimido[1,2-α]purine-10(3H)one (12), and N2-(2,6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (11). Adduct 9 has been previously identified in reactions of crotonaldehyde with DNA. However, the distribution of diastereomers was different in the acetaldehyde and crotonaldehyde reactions, indicating that the formation of 9 from acetaldehyde does not proceed through crotonaldehyde. Adduct 12 is an interstrand cross-link. Although previous evidence indicates the formation of cross-links in DNA reacted with acetaldehyde, this is the first reported structural characterization of such an adduct. This adduct is also found in crotonaldehyde-deoxyguanosine reactions, but in a diastereomeric ratio different than that observed here. A common intermediate, N2-(4-oxobut-2-yl)deoxyguanosine (6), is proposed to be involved in formation of adducts 9 and 12. Adduct 11 is produced ultimately from 3-hydroxybutanal, the major aldol condensation product of acetaldehyde. Levels of adducts 9, 11, and 12 were less than 10% of those of N2-ethylidenedeoxyguanosine (1) in reactions of acetaldehyde with DNA. As nucleosides, adducts 9, 11, and 12 were stable, whereas N2-ethylidenedeoxyguanosine (1) had a half-life of 5 min. These new stable adducts of acetaldehyde may be involved in determination of its mutagenic and carcinogenic properties.