In yeast a single type II DNA topoisomerase is involved in both the removal of DNA supercoils and the unlinking of intertwined pairs of newly replicated chromosomes or plasmids; in bacteria, two type II enzymes, DNA gyrase and DNA topoisomerase IV, function separately in the passage of DNA segments in cis and in trans. To deduce the molecular characteristics of these enzyme-mediated reactions, the efficiencies of supercoil removal and decatenation by the yeast enzyme upon the addition of a nonhydrolysable ATP analogue were determined. The probability that a bound enzyme transports a DNA segment in cis increases with positive or negative supercoiling of the DNA, and transport is nearly quantitative at high degrees of supercoiling. The relative probabilities of transporting a contiguous and noncontiguous DNA segment by a yeast enzyme bound to one member of a singly linked pair of 3.6-kb rings were calculated from the observed efficiency of decatenation. When the enzyme-bound ring is highly supercoiled, transport of a noncontiguous segment is more probable than a contiguous one. A DNA-bound yeast enzyme has no intrinsic bias in its selection of a contiguous or noncontiguous DNA segment for transport, and the selection is determined by DNA conformations. For the singly linked dimeric catenane studied, a high degree of supercoiling of the enzyme-bound DNA does not make supercoil removal more favourable than decatenation. In the case of bacterial gyrase, however, wrapping of a DNA segment around the enzyme is expected to strongly favour the transport of a contiguous segment.