The inhibition of restriction endonuclease cleavage by a series of bisquaternary ammonium derivatives (BQA-derivatives) which bind to the minor groove of DNA has been studied. The derivatives considered included six sequence-selective binders (SN 6570, SN 6999, SN 6050, SN 6132, SN 6131 and SN 18071) and four non-specific binders (SN 6113, SN 5754, SN 6324 and SN 4094) and can be distinguished by their activity on restriction endonucleases. Digestion experiments with pUC19 DNA were monitored electrophoretically using the transition of the covalently closed circular (ccc) DNA into the linear double stranded (lds) one. Only the sequence-specific binders inhibit the cleavage activity of restriction endonucleases EcoRI, SspI and DraI with four and six dAdT-base pairs within their restriction sites, while the activity of SalI and BamHI with less than four dAdT-sequences was unaffected. In contrast, the non-specific binding ligands were incapable of suppressing enzyme digestion. The inhibition of the restriction endonuclease PvuII indicates that ligand binding in close vicinity to the cleavage sites is also involved in the enzyme inhibition. The dAdT-content in proximity to the palindromic sequences of three DraI cutting sites in pUC19 DNA explains why the derivative SN 6053 protects these sequences in different manners. Gel shift experiments indicated that BQA-derivatives inhibit the DNA-enzyme complex formation if the ligand was added to the DNA before the enzyme. In contrast, complex formation between DNA and enzyme remained unchanged when the enzyme was added first.