The binding affinity of purified native Ustilago maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary structures was determined by gel shift and equilibrium binding analysis. Topoisomerase I exhibited cooperativity in binding to DNA regardless of the substrate structure. Further analysis demonstrated that cruciform DNA has two populations of binding sites for topoisomerase I while the other substrates (single-stranded DNA, DNA molecules containing six or one mismatched base pairs, hairpin, and fully homologous duplex DNA) have a single population of binding sites. The affinity of topoisomerase I for cruciform was found to be an order of magnitude higher affinity than for any of the other substrates. The high affinity of topoisomerase I for cruciform and specificity of topoisomerase I-cruciform structure interaction were confirmed by competition experiments. These studies demonstrate the high affinity of topoisomerase I for cruciform structure.