Abasic sites which are generated by hydrolysis of the glycosidic bonds connecting the heterocyclic nucleobases to the deoxyribose backbone represent the most common form of damage suffered by genomic DNA. Interestingly, an early report suggested that abasic sites can generate interstrand cross-links in duplex DNA, but the structure of these lesions has remained uncertain. The widespread occurrence of abasic sites in cellular DNA, along with the potent biological activities associated with cross-links, make it important to characterize interstrand cross-links derived from abasic sites in duplex DNA. Here, the results of gel electrophoretic and nanospray mass spectroscopic analyses of various DNA duplexes containing a single abasic site provide evidence that the aldehyde group of the DNA abasic site generates an interstrand cross-link via imine formation with the exocyclic N-2-amino group of a guanine residue on the opposing strand in 5'-d(CAp) sequences (where Ap is an abasic site).