Norfloxacin is a widely used antibacterial agent that inhibits DNA gyrase. This fluoroquinolone drug has significant interaction with double-stranded DNA, as judged from absorption and circular dichroism measurements. The mode of binding of norfloxacin to a variety of DNAs and polynucleotides has been investigated by electric linear dichroism. In the presence of calf thymus DNA, the drug chromophore is significantly tilted with respect to the DNA axis. This molecular arrangement contradicts classical intercalation. The orientation of the quinolone drug varies depending on the sequence of the target DNA. Binding to alternating copolymers is largely preferred compared to the corresponding homopolymers. The drug interacts preferentially with poly(dG-dC).(dG-dC) rather than with the other polynucleotides. The deletion of the 2-amino group of guanine (G-->I substitution) or the addition of a methyl group on cytosine residues (C-->methyl-C substitution) affect the drug-DNA interaction. The results show that norfloxacin is capable of interacting with a variety of DNA sequences, possibly via both minor and major groove contacts.