We examined the base specificity of the norfloxacin-DNA interaction by measuring the binding constant of norfloxacin to various synthetic polynucleotides, using the Stern-Volmer and the Benesi-Hildebrand methods. The equilibrium constants were largest for poly[d(G-C)2] and poly(dG).poly(dC), suggesting that norfloxacin binds preferentially to the G-C bases of calf thymus DNA. We also found that norfloxacin has a greater affinity for purine than for pyrimidine. The binding mode of norfloxacin to double-stranded polynucleotide was studied using circular and linear dichroism (CD and LD). When the norfloxacin was complexed to poly[d(G-C)2], poly(dG).poly(dC) and DNA, all of the complexes exhibited a similar weak, positive CD band and negative LD in the 300-350-nm region. A closer examination of the LD spectra suggests that the molecular plane of norfloxacin is near perpendicular relative to DNA helix axis that excludes the groove binding mode or surface binding of norfloxacin.