The interaction of the antibiotic drug norfloxacin with double-stranded DNA containing interior 5'-CpG-3', 5'-GpC-3', and 5'-GpG-3' steps was studied by 1H NMR. The drug is in fast exchange on the NMR timescale. A highly selective broadening of the imino proton resonances assigned to central CpG steps was observed after addition of drug, indicating an intercalation-like interaction. DNA sequences with central CpG steps also displayed broadening of non-hydrogen-bonded cytosine amino protons in the major groove upon addition of norfloxacin. Furthermore, a sequence-independent selective broadening of the adenine H2 resonance and an upfield shift of the guanine amino proton resonance, both protons located in the minor groove, was observed. Two-dimensional-NOESY spectra showed that no significant structural changes were induced in the DNA by the drug. The results suggest that the planar two-ring system of norfloxacin partially intercalates into CpG steps and that the drug also exhibits non-specific groove binding.