We compared various spectroscopic properties of a norfloxacin-single stranded DNA complex with those of norfloxacin-double stranded DNA complex. Norfloxacin binds to both double- and single stranded DNA, and we observed the following spectroscopic changes for both complexes: hypochromism in the norfloxacin absorption region in the absorption spectrum, the characteristic induced CD spectrum consisting of a weak positive band at 323 nm and a strong positive band at 280-300 nm followed by a negative band in the 260 nm region, a strong decrease in the fluorescence intensity and a red-shift in the fluorescence emission spectrum, and shorter fluorescence decay times. These results indicate that the environments of the bound norfloxacin in both DNAs are similar, although the equilibrium constant of the norfloxacin-single stranded DNA was twice as high as the norfloxacin-double stranded DNA complex. Both complexes were thermodynamically favored with similar negative ΔGo. Negative ΔHo terms contribute to these spontaneous reactions; ΔSo term was unfavorable.