The interaction of DNA topoisomerase I (topo I) with a set of single- and double-stranded oligonucleotides containing 5-27 mononucleotides was investigated. All single- and double-stranded oligonucleotides were found to inhibit competitively the supercoiled DNA relaxation reaction catalyzed by topo I. The enzyme affinity for specific sequence pentanucleotides of the scissile (GACTT, Ki = 2 microM) and non-cleaved chain (AAGTC, Ki = 110 microM) is about 2-4 orders of magnitude higher than that for non-specific oligonucleotides. This specific sequence affinity increases in several cases; lengthening of single-stranded oligonucleotides, formation of stable duplexes between complementary oligonucleotides and preincubation of the enzyme with ligands before addition of supercoiled DNA. We assume that oligonucleotides having a high affinity to the enzyme can offer a unique opportunity for rational design of topoisomerase-targeting drugs.