Two series of tethered nitrogen mustards based on the minor groove-binding and A/T sequence-specific natural product distamycin have been synthesized and evaluated. The conjugates, which have a modified dimethylamino C-terminus, are comprised of one, two or three pyrrole carboxamide units linked to either benzoic acid mustard (BAM) or chlorambucil (CHL). The DNA binding properties, in vitro cytotoxicities and DNA cross-linking abilities were determined for each of the conjugates. The conjugates were found to bind preferentially to poly(dA.dT) compared to poly(dG.dC) DNA by ethidium displacement and circular dichroism. The di- and tripyrrole conjugates had higher binding affinities than the monopyrrole conjugates. All the conjugates were more cytotoxic than the nitrogen mustards themselves. Cytotoxicity increased with the increase from one to three pyrrole units and the CHL conjugates were more cytotoxic than the corresponding BAM analogues. The CHL conjugates were able to cross-link plasmid DNA at a 10-fold lower dose than CHL itself. The BAM conjugates showed < 10% cross-linking at doses which gave 100% cross-linking with the CHL conjugates. In cells, the CHL conjugates showed significant cross-linking at the IC50 values, while the BAM conjugates showed no evidence of cross-link formation even at 10 times the IC50 value. These results are discussed in reference to a series of previously reported GC-recognizing imidazole analogues possessing the same nitrogen mustard groups.