trans-4-Hydroxynonenal (HNE) is a peroxidation product of ω-6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1,N2-dG adducts. The adduct of (6S,8R,11S) stereochemistry forms inter-strand N2-dG:N2-dG cross-links in the 5′-CpG-3′ sequence. It has been compared with the (6R,8S,11R) adduct, incorporated into 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTCGCTAGC)-3′, containing the 5′-CpG-3′ sequence (X = HNE-dG). Both adducts rearrange in DNA to N2-dG aldehydes. These aldehydes exist in equilibrium with diastereomeric cyclic hemiacetals, in which the latter predominate at equilibrium. These cyclic hemiacetals mask the aldehydes, explaining why DNA cross-linking is slow compared to related 1,N2-dG adducts formed by acrolein and crotonaldehyde. Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals are located within the minor groove. However, the (6S,8R,11S) cyclic hemiacetal orients in the 5′-direction, while the (6R,8S,11R) cyclic hemiacetal orients in the 3′-direction. The conformations of the diastereomeric N2-dG aldehydes, which are the reactive species involved in DNA cross-link formation, have been calculated using molecular mechanics methods. The (6S,8R,11S) aldehyde orients in the 5′-direction, while the (6R,8S,11R) aldehyde orients in the 3′-direction. This suggests a kinetic basis to explain, in part, why the (6S,8R,11S) HNE adduct forms interchain cross-links in the 5′-CpG-3′ sequence, whereas (6R,8S,11R) HNE adduct does not. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease.