cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana lambdagt11 and lambdaFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOP1 probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody.