The nucleotide sequence specificity of the DNA interstrand cross-linking reaction of 2,5-bis(1-aziridinyl)1,4-benzoquinone (DZQ) and the covalent structure of the predominant cross-link were determined in synthetic DNA duplexes. A panel of synthetic DNAs was exposed to aqueous DZQ and analyzed for interstrand cross-links by denaturing polyacrylamide gel electrophoresis (DPAGE). Those containing the sequences 5’-d(GGCC), 5’-d(GGGCCC), and 5’-d(GGGGC) afforded relatively abundant interstrand cross-linked products. Exposure of some of these DZQ-cross-linked DNAs to hot aqueous piperidine followed by DPAGE analysis afforded DNA strand scission products with mobility identical to that of the products of the Maxam-Gilbert G reaction, consistent with a dG-to-dG nucleotide connectivity at the duplex sequences 5’-d(GN(n)C), n = 0, 1, and 2, with some preference for n = 1. The covalent structure of the cross-link was assigned by a combination of spectroscopic measurements and chemical reactions as bridging N-7 atoms of dG residues on opposite strands of DNA through an intervening 2,5-bis(ethyleneamino)-1,4-benzoquinone unit. The origin of the nucleotide sequence preference of DZQ is discussed.