The activity of DAPI, on a number of DNA-directed enzymes involved in DNA topology, transcription, replication and repair, is reported in this paper. DAPI was always more inhibitory than ethidium bromide, in particular against RNA polymerase and DNA ligase, which seemed to be specifically affected. While the effect on RNA polymerase is likely due to a preferential occupancy of the promoter region, that on DNA ligase could rely upon a mechanism of steric hindrance in the minor groove. These phenomena, independently from an alteration of the tertiary structure of DNA by the ligand, can account for the previously reported inhibition of plasmid expression in Escherichia coli.