AbstractA melting experiment was performed on the whole set of populations of the replicative form of ϕX174 DNA, which can be obtained treating this DNA with rat liver nicking‐closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double‐stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double‐stranded DNA: for native ϕX174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpr