DNA relaxation catalysed by topoisomerase I is based on the reversible DNA cleavage. The reaction is inhibited by binding of splicing protein SF2/ASF. a substrate for the kinase activity of topoisomerase, I. In this paper. we show a novel binding site forSF2/ ASF in the cap region of topoisomerase I (amino acids 215 433) which interacts with the region containing two closely spaced RRM domains of SF2/ASF (amino acids 1-194). The sites were defined by a Set of pull down experiments with isolated recombinant polypeptides. We also indicate that the novel site is responsible for the inhibition of DNA cleavage. The polypeptide containing tandem RRM domains inhibited DNA cleavage by topoisomerase I similarly as the complete SF2/ASF. Moreover, interaction between the tandem RRM domains and the cap region was not possible in the presence of DNA. © 2005 Elsevier Inc. All rights reserved.