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Sedimentation velocity techniques have been used to study the effect of the antibiotic drug netropsin (Nt) on the structural properties of different forms of closed circular DNA. Nt removes supercoils upon interaction with positively supercoiled DNA and introduces negative supercoils upon binding to relaxed DNA. Applying the classic assay of DNA unwinding by ethidium to complexes of negatively supercoiled DNA with Nt, we found that, compared with naked DNA, more ethidium is needed to convert the Nt-DNA complex into a superhelix-free form. From the shift in the critical ethidium/nucleotide binding ratio, a Nt-mediated decrease in the linking number difference and superhelix density (or increase in the number of superhelical turns) of negatively supercoiled DNA is calculated, synonymous with an increase in the linking number and a decrease in the helical repeat of the relaxed form. A significant influence of the presence of intrinsic bends in the DNA duplex on the magnitude of the drug-induced alterations in DNA supercoiling could not be detected. Although it is unclear whether the primary action of netropsin consists of changes in writhe or twist of DNA, or both, the measurable effects can be described by a Nt-induced overwinding of the DNA doulbe helix. Nt-specific DNA overwinding angels can also be derived from the (positive) linking number difference observed in literature upon relaxation of supercoiled Nt-DNA complexes by topoisomerase I and subsequent removal of Nt. A compilation of the data available from literature and our own work shows that the average Nt-mediated DNA overwinding angle exhibits a strong decrease with increasing Nt/nucleotide ratio. This finding suggests that Nt exerts the strongest effects on DNA supercoiling when interacting with DNA binding sites which have the highest affinity and, hence, primarily are occupied by Nt. [ABSTRACT FROM AUTHOR]