Norfloxacin, a quinolone antibacterial reagent, has been studied with respect to its binding to calf thymus DNA using fluorescence and linear dichroism techniques and unwinding of supercoiled DNA. The fluorescence of norfloxacin is strongly quenched in the presence of DNA and using this decrease in a fluorescence titration the equilibrium constant of the complex formation was established to be 2.8 x 10(3) M-1. The electric transition moments of the norfloxacin chromophore have been analyzed using fluorescence anisotropy, magnetic circular dichroism, and linear dichroism in stretched poly(vinyl alcohol) film and INDO/S calculations. These data are then used to interpret flow linear dichroism results for the norfloxacin-DNA complex. The transition moments for the long-wavelength transitions are found to be oriented at about 65.0-85.0 degrees with respect to the DNA helix axis. A near perpendicular orientation of the norfloxacin chromophore plane makes it possible to exclude classical groove or surface binding modes. The possibility of a classical intercalation binding mode also can be ruled out from unwinding experiments. However, it is shown that the molecular plane of norfloxacin is near perpendicular relative to the DNA helix axis with a possibility of a bending of the DNA helix at the binding site.