The mustard derivative, bis-N,N'-[3-(N-(2-chloroethyl)-N-ethyl)amino-5- [N,N-dimethylamino)methyl)aminophenyl]-1,4-benzenedicarboxamide has been synthesized from 3-acetamido-5-nitrobenzoic acid in a 6-step procedure. This compound alkylates exclusively in the minor groove of DNA, at the N3 site of adenines occurring in sequences of runs of adenines and (to a small extent) at 5'-TA and 5'-AT sites. Gel electrophoresis studies and in vitro cytotoxicity assays against repair-deficient AA8 mutant cell lines show it has a high degree of DNA interstrand cross-linking ability.