The interaction of anti-tumour drugs with reconstituted chromatin has been investigated using defined nucleosomal complexes. This allowed the effect of nucleosome cores on drug-induced DNA damage to be assessed for four nitrogen mustard analogues, dimethylsulphate and three cisplatin analogues. A defined nucleosomal complex was employed that contained two precisely positioned nucleosome cores. The construct was then subjected to drug treatment, and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At the sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug–DNA interactions. Results were combined with previous data obtained for other DNA-damaging drugs investigated using the same nucleosomal construct. For most of the DNA-damaging agents studied, this method revealed protection at the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and revealed differences between the examined compounds. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA and were impeded from damaging nucleosomal core DNA. In contrast, smaller molecules had greater access to nucleosomal core DNA.