As part of an investigation into the sequence selectivity of the nogalamycin-DNA interaction, the 1.58 Å structure of nogalamycin complexed with d 5 ' (TGTACA) 2 has been determined by single-crystal X-ray analysis. The complex crystallised in the orthorhombic space groupP2 1 2 1 2 1 with cell dimensionsa=26.3 Å,b=52.0 Å andc=67.1 Å, incor- porating twoB-DNA duplexes and four nogalamycin molecules in the asymmetric unit. The final refined structure included 97 water molecules, one spermine molecule, two acetate ions and one sodium ion, yielding an overallRfactor of 19.2% (calculated using all 12,358 reflections in the resolution range 10.7 to 1.6 Å) and anR f r e e of 23.7% (using 1229 test reflections).The d 5 ' (TGTACA) 2 sequence was designed to include the d 5 ' (TpG) pyrimidine-purine base step that has been ascertained as a preferential intercalation site. The complexes in the asymmetric unit are globally similar; one nogalamycin molecule intercalates between each d 5 ' (TpG) step in each duplex. The DNA of each complex exists as a distortedB-DNA duplex displaying someZ-DNA character in the form of C3'endosugars at some residues. Structural comparisons between the d 5 ' (TGTACA) 2 - nogalamycin 2 complex and the complexes of this drug with the sequences d 5 ' (TGATCA) 2 and d 5 ' ( 5 M e CGT(pS)A 5 M e CG) 2 highlight differences in binding interactions between nogalamycin and these various triplet DNA binding sites, with regards to the stability of drug intercalation, which in turn is correlated to effective levels of cytotoxicity towards tumour cells. The number of both direct and water-mediated hydrogen bonds and van der Waal's interactions between substituents of nogalamycin and the d 5 ' (TGTACA) 2 and d 5 ' ( 5 M e CGT(pS)A 5 M e CG) 2 sequences are significantly greater than those made with the d 5 ' (TGATCA) 2 sequence, suggesting that the central d 5 ' (TpA) in the former confers additional stability to the complex once the drug has bound.