Bizelesin is the first anticancer drug capable of damaging specific regions of the genome with clusters of its binding sites T(A/T)(4)A. This study characterized the sequence- and region-specificity of a bizelesin analogue, U-78779, designed to interact with mixed A/T-G/C motifs. At the nucleotide level, U-78779 was found to prefer runs of A/Ts interspersed with 1 or 2 G/C pairs, although 25% of the identified sites corresponded to pure AT motifs similar to bizelesin sites. The in silico computational analysis showed that the preferred mixed A/T-G/C motifs distribute uniformly at the genomic level. In contrast, the secondary, pure AT motifs (A/T)(6)A were found densely clustered in the same long islands of AT-rich DNA that bizelesin targets. Mapping the sites and quantitating the frequencies of U-78779 adducts in model AT island and non-AT island naked DNAs demonstrated that clusters of pure AT motifs outcompete isolated mixed A/T-G/C sites in attracting drug binding. Regional preference of U-78779 for AT island domains was verified also in DNA from drug-treated cells. Thus, while the primary sequence preference gives rise to non-region-specific scattered lesions, the clustering of the minor pure AT binding motifs seems to determine region-specificity of U-78779 in the human genome. The closely correlated cytotoxic activities of U-78779 and bizelesin in several cell lines further imply that both drugs may share common cellular targets. This study underscores the significance of the genome factor in a drug's potential for region-specific DNA damage, by showing that it can take precedence over drug binding preferences at the nucleotide level.